Team:Duke/Notebook/July

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<td colspan="7"> May 2014 </td>
 
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<td colspan="7" align=center><i> Month 2 of Project </i></td>
 
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<td>18</td>
 
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<td>25</td>
 
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<td>26</td>
 
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<td>27</td>
 
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<td>28</td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#may29">29</a></td>
 
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<td>30</td>
 
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<td>31</td>
 
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<td colspan="7"> June 2014 </td>
 
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<td colspan="7" align=center><i> Month 3 of Project </i></td>
 
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<td>sun</td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun1">1</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun2">2</a></td>
 
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<td>3</td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun4">4</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun5">5</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun6">6</a></td>
 
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<td>7</td>
 
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<td>8</td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun9">9</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun10">10</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun11">11</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun12">12</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun13">13</a></td>
 
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<td>14</td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun15">15</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun16">16</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun17">17</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun18">18</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun19">19</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun20">20</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun21">21</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun22">22</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun23">23</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun24">24</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun25">25</a></td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun26">26</a></td>
 
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<td>27</td>
 
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<td>28</td>
 
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<tr>
 
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<td>29</td>
 
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<td><a href="https://2014.igem.org/Team:Duke/Notebook#jun30">30</a></td>
 
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<td></td>
 
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<div id="rightnb">
 
<div class="day">
<div class="day">
<a id="jul1"><h2> July 1 </h2></a>
<a id="jul1"><h2> July 1 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: make LB+Cm plates </span></p><ul class="c7 lst-kix_wg1rv7zn1r0-0 start"><li class="c1"><span class="c3">Four sleeves of plates with 4x500mL medium</span></li><li class="c1"><span class="c3">34mg/mL chloramphenicol</span></li></ul>
+
<p class="obj">Objective: make LB+Cm plates </p>
-
<p class="obj"><span class="c3">Objective: insert crRNAs into pdCas9 and scaffold</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Plate results</span></p><ul class="c7 lst-kix_rlhu7vd12z5-0 start"><li class="c1"><span>Experimental had lots of colonies, but backbone-only control did as well</span></li></ul><ul class="c7 lst-kix_rlhu7vd12z5-1 start"><li class="c2"><span>reasoning: similarity of BsaI overhangs may lead to re-ligation</span></li><li class="c2"><span>solution: treat with CIP before ligation</span></li></ul><p class="c6 c16"><span class="c4">Agarose gel of BsaI cuts of pdCas9 and Repeat scaffold </span></p><ul class="c7 lst-kix_lqrhjh6f7a91-0 start"><li class="c1"><span>plasmids used in 6/30/14 transformation</span></li><li class="c1"><span>to make sure BsaI is cutting properly</span></li><li class="c1"><span class="c4">Results: single band in both appears to be cut DNA</span></li></ul>
+
<ul>
 +
<li>Four sleeves of plates with 4x500mL medium</li>
 +
<li>34mg/mL chloramphenicol</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: insert crRNAs into pdCas9 and scaffold</p>
 +
<p>Plate results</p>
 +
 
 +
<ul>
 +
<li>Experimental had lots of colonies, but backbone-only control did as well</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>reasoning: similarity of BsaI overhangs may lead to re-ligation</li>
 +
<li>solution: treat with CIP before ligation</li>
 +
</ul>
 +
<p>Agarose gel of BsaI cuts of pdCas9 and Repeat scaffold </p>
 +
 
 +
<ul>
 +
<li>plasmids used in 6/30/14 transformation</li>
 +
<li>to make sure BsaI is cutting properly</li>
 +
<li>Results: single band in both appears to be cut DNA</li>
 +
</ul>
<!-- https://2014.igem.org/File:GEL_MCF_7-1-14.png or https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png-->
<!-- https://2014.igem.org/File:GEL_MCF_7-1-14.png or https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png-->
<a href="https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png"></a>
<a href="https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e8/GEL_MCF_7-1-14.png"></a>
-
<p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">CIP treatment of pdCas9 and Repeat scaffold (both BsaI cut)</span></p><ul class="c7 lst-kix_7ok3mhafto22-0 start"><li class="c1"><span>pdCas9: 10uL plasmid in 20 uL reaction with 1.5uL CIP</span></li><li class="c1"><span>Repeat scaffold: 22uL plasmid in 30 uL reaction with 2 uL CIP</span></li><li class="c1"><span>2 hours at 37C (longer than standard protocol)</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">PCR cleanup of pdCas9 and Repeat scaffold CIP treatments</span></p><ul class="c7 lst-kix_gew6d09gxhbp-0 start"><li class="c1"><span>Final concentrations too low to be of use</span></li></ul><ul class="c7 lst-kix_gew6d09gxhbp-1 start"><li class="c2"><span>Particularly negligible in pdCas9</span></li><li class="c2"><span>Need to culture and cut more plasmid to try ligation again</span></li></ul><p class="c6 c16"><span class="c4">Spread plates from pdCas9 and Repeat scaffold frozen stocks</span></p><ul class="c7 lst-kix_z57kxb2fodhm-0 start"><li class="c1"><span>Used tube &ldquo;1&rdquo; for repeat scaffold</span></li><li class="c1"><span>Plated on fresh LB+Cm plates</span></li></ul><ul class="c7 lst-kix_z57kxb2fodhm-1 start"><li class="c2"><span>Plates still slightly wet, difficult to spread evenly</span></li></ul>
+
<p>CIP treatment of pdCas9 and Repeat scaffold (both BsaI cut)</p>
-
<p class="obj"><span class="c3">Objective: make CCEC stock of DH5alpha-ZI strain</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Diluted back 5mL culture in morning</span></p><ul class="c7 lst-kix_ero6g17ifbi-0 start"><li class="c1"><span>1mL into 5mL LB+Spec</span></li></ul><ul class="c7 lst-kix_ero6g17ifbi-1 start"><li class="c2"><span>note for future: Spec not necessary for most procedures, chromosomally integrated genes are more stable</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Inoculated 1 mL culture into each of two 250mL flasks</span></p><ul class="c7 lst-kix_1dhg46gcjq2a-0 start"><li class="c1"><span>Flasks contain SOC</span></li><li class="c1"><span>30C shaker for overnight growing</span></li></ul>
+
<ul>
 +
<li>pdCas9: 10uL plasmid in 20 uL reaction with 1.5uL CIP</li>
 +
<li>Repeat scaffold: 22uL plasmid in 30 uL reaction with 2 uL CIP</li>
 +
<li>2 hours at 37C (longer than standard protocol)</li>
 +
</ul>
 +
 
 +
<p>PCR cleanup of pdCas9 and Repeat scaffold CIP treatments</p>
 +
 
 +
<ul>
 +
<li>Final concentrations too low to be of use</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Particularly negligible in pdCas9</li>
 +
<li>Need to culture and cut more plasmid to try ligation again</li>
 +
</ul>
 +
<p>Spread plates from pdCas9 and Repeat scaffold frozen stocks</p>
 +
 
 +
<ul>
 +
<li>Used tube &ldquo;1&rdquo; for repeat scaffold</li>
 +
<li>Plated on fresh LB+Cm plates</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Plates still slightly wet, difficult to spread evenly</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: make CCEC stock of DH5alpha-ZI strain</p>
 +
<p>Diluted back 5mL culture in morning</p>
 +
 
 +
<ul>
 +
<li>1mL into 5mL LB+Spec</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>note for future: Spec not necessary for most procedures, chromosomally integrated genes are more stable</li>
 +
</ul>
 +
<p>Inoculated 1 mL culture into each of two 250mL flasks</p>
 +
 
 +
<ul>
 +
<li>Flasks contain SOC</li>
 +
<li>30C shaker for overnight growing</li>
 +
</ul>
</div>
</div>
</div>
</div>
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<a id="jul2"><h2> July 2 </h2></a>
<a id="jul2"><h2> July 2 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: Make CCEC stock of DH5alpha-ZI strain</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Made fresh CCMB-80 buffer stock</span></p><ul class="c7 lst-kix_t9k6okhjl9gq-0 start"><li class="c1"><span>precipitate formed during pH adjusting and may have filtered out</span></li></ul><ul class="c7 lst-kix_t9k6okhjl9gq-1 start"><li class="c2"><span>could be detrimental to final product</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Followed iGEM online protocol to make CCEC from Z1 strain</span></p><ul class="c7 lst-kix_q2gw67s9hqh2-0 start"><li class="c1"><span>Overnight culture was much more concentrated than recommended</span></li></ul><ul class="c7 lst-kix_q2gw67s9hqh2-1 start"><li class="c2"><span>OD &gt;1</span></li><li class="c2"><span>Did not have enough SOC to dilute back fully, so diluted back only a bit</span></li></ul><ul class="c7 lst-kix_vfxch5kb8uzf-0 start"><li class="c1"><span>Had to vortex to resuspend cells at two stages of procedure</span></li><li class="c1"><span>Final OD after procedure ~1.6 for 100mL of cells</span></li><li class="c1"><span>Aliquoted 1mL into tubes, froze at -80C</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Next steps: </span></p><ul class="c7 lst-kix_aadq3fbhgjrz-0 start"><li class="c1"><span>Test competence with useful strains containing Lac, Tet promoters</span></li></ul>
+
<p class="obj">Objective: Make CCEC stock of DH5alpha-ZI strain</p>
 +
<p>Made fresh CCMB-80 buffer stock</p>
-
<p class="obj"><span class="c3">Objective: insert crRNAs into pdCas9 and Repeat-scaffold</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Culture colonies from plates of pdCas9 and pSB1C3-Repeat-scaffold</span></p><ul class="c7 lst-kix_p8kxtmebz969-0 start"><li class="c1"><span>3 colonies per plate in LB+Cm medium at 37C overnight</span></li><li class="c1"><span>Plates were messy--hard to get individual colonies </span></li></ul>
+
<ul>
 +
 
 +
<li>precipitate formed during pH adjusting and may have filtered out</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>could be detrimental to final product</li>
 +
</ul>
 +
<p>Followed iGEM online protocol to make CCEC from Z1 strain</p>
 +
 
 +
<ul>
 +
<li>Overnight culture was much more concentrated than recommended</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>OD &gt;1</li>
 +
<li>Did not have enough SOC to dilute back fully, so diluted back only a bit</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Had to vortex to resuspend cells at two stages of procedure</li>
 +
<li>Final OD after procedure ~1.6 for 100mL of cells</li>
 +
<li>Aliquoted 1mL into tubes, froze at -80C</li>
 +
</ul>
 +
<p>Next steps: </p>
 +
 
 +
<ul>
 +
<li>Test competence with useful strains containing Lac, Tet promoters</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: insert crRNAs into pdCas9 and Repeat-scaffold</p>
 +
<p>Culture colonies from plates of pdCas9 and pSB1C3-Repeat-scaffold</p>
 +
 
 +
<ul>
 +
<li>3 colonies per plate in LB+Cm medium at 37C overnight</li>
 +
<li>Plates were messy--hard to get individual colonies </li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 183: Line 136:
<a id="jul3"><h2> July 3 </h2></a>
<a id="jul3"><h2> July 3 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: insert crRNAs into pdCas9 and Repeat-scaffold</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Miniprep cultures from 7/2</span></p><ul class="c7 lst-kix_55qfcnduv952-0 start"><li class="c1"><span>Three tubes of pdCas9 with concentrations 143.4, 145.7, 151.4 ng/uL</span></li><li class="c1"><span>Three tubes of Repeat-scaffold with concentrations 166.6, 145.8, 159.6 ng/uL</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Digest tubes 1 and 2 of pdCas9 and tubes 1 and 2 of Repeat-scaffold</span></p><ul class="c7 lst-kix_fd86mb3nli0h-0 start"><li class="c1"><span>to insert crRNAs</span></li><li class="c1"><span>Digest with BsaI in cutsmart buffer</span></li><li class="c1"><span>50 uL DNA in 100 uL reactions with 7.5 uL BsaI</span></li><li class="c1"><span>3 hours 20 min at 37C</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">PCR cleanup of BsaI digest</span></p><ul class="c7 lst-kix_fn8o106jg3tb-0 start"><li class="c1"><span>final concentrations (in 30 uL each):</span></li></ul><ul class="c7 lst-kix_fn8o106jg3tb-1 start"><li class="c2"><span>pdCas9: 65.4, 70.5 ng/uL</span></li><li class="c2"><span>Repeat-scaffold: 122.3, 114.8 ng/uL</span></li></ul><p class="c6 c16"><span class="c4">CIP treatment of pdCas9 and Repeat-scaffold BsaI digests</span></p><ul class="c7 lst-kix_e6lalg2ram87-0 start"><li class="c1"><span>Two digest tubes pooled into one CIP tube for each backbone</span></li><li class="c1"><span>60 uL DNA in 100 uL reactions with 5 uL CIP in cutsmart buffer</span></li><li class="c1"><span>1.5 hours at 37C</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">PCR cleanup of CIP treatment</span></p><ul class="c7 lst-kix_1nkvpajg830x-0 start"><li class="c1"><span>final concentrations (in 30 uL):</span></li></ul><ul class="c7 lst-kix_1nkvpajg830x-1 start"><li class="c2"><span>pdCas9: 32.5 ng/uL</span></li><li class="c2"><span>Repeat scaffold 129.9 ng/uL</span></li></ul><p class="c6 c16"><span class="c4">Annealed oligos of crRNA inserts for ligation</span></p><ul class="c7 lst-kix_2aswvyon22br-0 start"><li class="c1"><span>3 crRNAs (GFP1, GFP2, GFP3) with 2 oligos each</span></li><li class="c1"><span>5uL each oligo heated to ~95C, slowly brought to rt in water bath</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Ligation of crRNA inserts into pdCas9 and Repeat-scaffold</span></p><ul class="c7 lst-kix_jnn5awdyyykz-0 start"><li class="c1"><span>6 working tubes + 5 controls</span></li></ul><ul class="c7 lst-kix_jnn5awdyyykz-1 start"><li class="c2"><span>pdCas9 + each crRNA (GFP1, GFP2, or GFP3)</span></li><li class="c2"><span>Repeat-scaffold + each crRNA (GFP1, GFP2, or GFP3)</span></li><li class="c2"><span>pdCas9 Backbone only control</span></li><li class="c2"><span>Repeat-scaffold Backbone only control</span></li><li class="c2"><span>Insert only controls for each insert</span></li></ul><ul class="c7 lst-kix_jnn5awdyyykz-0"><li class="c1"><span>100ng = 3uL pdCas9, BsaI digested and CIP treated and cleaned</span></li><li class="c1"><span>100ng = 0.8uL Repeat-scaffold, BsaI digested and CIP treated and cleaned</span></li><li class="c1"><span>1uL annealed oligo insert</span></li><li class="c1"><span>10 uL reactions with 0.5 uL T4 Ligase</span></li><li class="c1"><span>Transformed into DH5alpha and plated on LB+Cm</span></li></ul>
+
<p class="obj">Objective: insert crRNAs into pdCas9 and Repeat-scaffold</p>
 +
<p>Miniprep cultures from 7/2</p>
-
<p class="obj"><span class="c3">Objective: Test competency and effectiveness of DH5alpha-Z1 strain</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">CCEC Transformation efficiency test</span></p><ul class="c7 lst-kix_c8mfx2aowjtj-0 start"><li class="c1"><span>Transformed 5 tubes of pSB1C3-K741002 into DH5alpha-Z1 CCEC</span></li></ul><ul class="c7 lst-kix_c8mfx2aowjtj-1 start"><li class="c2"><span>Total amount of DNA in 10 uL: 130ng, 13ng, 1.3ng, 0.13ng, and 0.013ng</span></li></ul><ul class="c7 lst-kix_c8mfx2aowjtj-0"><li class="c1"><span>Also for use in testing pLac regulation in Z1 strain</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Transformed iGEM kit plate 3-6G into DH5alpha-Z1</span></p><ul class="c7 lst-kix_msvgjfrjqc6j-0 start"><li class="c1"><span>contains pSB1C3-BBa_I13521: pTet-RBS-mRFP</span></li><li class="c1"><span>in order to test regulation of pTet in Z1 strain</span></li></ul>
+
<ul>
 +
<li>Three tubes of pdCas9 with concentrations 143.4, 145.7, 151.4 ng/uL</li>
 +
<li>Three tubes of Repeat-scaffold with concentrations 166.6, 145.8, 159.6 ng/uL</li>
 +
</ul>
 +
<p>Digest tubes 1 and 2 of pdCas9 and tubes 1 and 2 of Repeat-scaffold</p>
 +
 
 +
<ul>
 +
<li>to insert crRNAs</li>
 +
<li>Digest with BsaI in cutsmart buffer</li>
 +
<li>50 uL DNA in 100 uL reactions with 7.5 uL BsaI</li>
 +
<li>3 hours 20 min at 37C</li>
 +
</ul>
 +
<p>PCR cleanup of BsaI digest</p>
 +
 
 +
<ul>
 +
<li>final concentrations (in 30 uL each):</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pdCas9: 65.4, 70.5 ng/uL</li>
 +
<li>Repeat-scaffold: 122.3, 114.8 ng/uL</li>
 +
</ul>
 +
<p>CIP treatment of pdCas9 and Repeat-scaffold BsaI digests</p>
 +
 
 +
<ul>
 +
<li>Two digest tubes pooled into one CIP tube for each backbone</li>
 +
<li>60 uL DNA in 100 uL reactions with 5 uL CIP in cutsmart buffer</li>
 +
<li>1.5 hours at 37C</li>
 +
</ul>
 +
<p>PCR cleanup of CIP treatment</p>
 +
 
 +
<ul>
 +
<li>final concentrations (in 30 uL):</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pdCas9: 32.5 ng/uL</li>
 +
<li>Repeat scaffold 129.9 ng/uL</li>
 +
</ul>
 +
<p>Annealed oligos of crRNA inserts for ligation</p>
 +
 
 +
<ul>
 +
<li>3 crRNAs (GFP1, GFP2, GFP3) with 2 oligos each</li>
 +
<li>5uL each oligo heated to ~95C, slowly brought to rt in water bath</li>
 +
</ul>
 +
<p>Ligation of crRNA inserts into pdCas9 and Repeat-scaffold</p>
 +
 
 +
<ul>
 +
<li>6 working tubes + 5 controls</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pdCas9 + each crRNA (GFP1, GFP2, or GFP3)</li>
 +
<li>Repeat-scaffold + each crRNA (GFP1, GFP2, or GFP3)</li>
 +
<li>pdCas9 Backbone only control</li>
 +
<li>Repeat-scaffold Backbone only control</li>
 +
<li>Insert only controls for each insert</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>100ng = 3uL pdCas9, BsaI digested and CIP treated and cleaned</li>
 +
<li>100ng = 0.8uL Repeat-scaffold, BsaI digested and CIP treated and cleaned</li>
 +
<li>1uL annealed oligo insert</li>
 +
<li>10 uL reactions with 0.5 uL T4 Ligase</li>
 +
<li>Transformed into DH5alpha and plated on LB+Cm</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Test competency and effectiveness of DH5alpha-Z1 strain</p>
 +
<p>CCEC Transformation efficiency test</p>
 +
 
 +
<ul>
 +
<li>Transformed 5 tubes of pSB1C3-K741002 into DH5alpha-Z1 CCEC</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Total amount of DNA in 10 uL: 130ng, 13ng, 1.3ng, 0.13ng, and 0.013ng</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Also for use in testing pLac regulation in Z1 strain</li>
 +
</ul>
 +
<p>Transformed iGEM kit plate 3-6G into DH5alpha-Z1</p>
 +
 
 +
<ul>
 +
<li>contains pSB1C3-BBa_I13521: pTet-RBS-mRFP</li>
 +
<li>in order to test regulation of pTet in Z1 strain</li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 192: Line 232:
<a id="jul4"><h2> July 4 </h2></a>
<a id="jul4"><h2> July 4 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c0 c3">Objective: ligate crRNAs into pdCas9 and Repeat-scaffold</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Plate results for ligation of crRNA GFP1, GFP2, and GFP3 into two backbones:</span></p><ul class="c7 lst-kix_iazs54ynmo4k-0 start"><li class="c1"><span class="c0">~1000 colonies on all experimental plates</span></li><li class="c1"><span class="c0">BO control for pdCas9 has ~500 colonies</span></li><li class="c1"><span class="c0">BO control for Repeat scaffold has ~1000 colonies</span></li><li class="c1"><span class="c0">IO controls all have 0 colonies</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Cultured 4 copies of each crRNA-GFP1,2,3 in pdCas9 backbone</span></p><ul class="c7 lst-kix_aej47evtc2n3-0 start"><li class="c1"><span class="c0">12 tubes total in LB+Cm</span></li><li class="c1"><span class="c0">None from Repeat-scaffold attempts</span></li></ul>
+
<p class="obj">Objective: ligate crRNAs into pdCas9 and Repeat-scaffold</p>
 +
<p>Plate results for ligation of crRNA GFP1, GFP2, and GFP3 into two backbones:</p>
-
<p class="obj"><span class="c0 c3">Objective: Test competency and effectiveness of DH5alpha-Z1 strain</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">CCEC Test plate results:</span></p><ul class="c7 lst-kix_b4hk2o9jds1l-0 start"><li class="c1"><span class="c0">130 ng: 100s of colonies</span></li><li class="c1"><span class="c0">13 ng:~100 colonies</span></li><li class="c1"><span class="c0">1.3 ng: ~20 colonies</span></li><li class="c1"><span class="c0">0.13 ng: 0 colonies</span></li><li class="c1"><span class="c0">0.013 ng: 0 colonies</span></li><li class="c1"><span class="c0 c4">Competency ~10 colonies/ng (low but working)</span></li></ul><p class="c6"><span class="c0 c4">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Results of transformation of pSB1C3-BBa_I13521 into DH5alpha-Z1</span></p><ul class="c7 lst-kix_c6zx83gas5j4-0 start"><li class="c1"><span class="c0">Plate had ~5 colonies</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Cultured colonies for Tet and Lac testing in DH5alpha-Z1</span></p><ul class="c7 lst-kix_r3j2h2jrv2rl-0 start"><li class="c1"><span class="c0">One colony of pSB1C3-K741002</span></li><li class="c1"><span class="c0">One colony of pSB1C3-BBa_I13521</span></li><li class="c1"><span class="c0">In LB+Cm</span></li></ul>
+
<ul>
 +
<li>~1000 colonies on all experimental plates</li>
 +
<li>BO control for pdCas9 has ~500 colonies</li>
 +
<li>BO control for Repeat scaffold has ~1000 colonies</li>
 +
<li>IO controls all have 0 colonies</li>
 +
</ul>
 +
<p>Cultured 4 copies of each crRNA-GFP1,2,3 in pdCas9 backbone</p>
 +
 
 +
<ul>
 +
<li>12 tubes total in LB+Cm</li>
 +
<li>None from Repeat-scaffold attempts</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Test competency and effectiveness of DH5alpha-Z1 strain</p>
 +
<p>CCEC Test plate results:</p>
 +
 
 +
<ul>
 +
<li>130 ng: 100s of colonies</li>
 +
<li>13 ng:~100 colonies</li>
 +
<li>1.3 ng: ~20 colonies</li>
 +
<li>0.13 ng: 0 colonies</li>
 +
<li>0.013 ng: 0 colonies</li>
 +
<li>Competency ~10 colonies/ng (low but working)</li>
 +
</ul>
 +
<p>Results of transformation of pSB1C3-BBa_I13521 into DH5alpha-Z1</p>
 +
 
 +
<ul>
 +
<li>Plate had ~5 colonies</li>
 +
</ul>
 +
<p>Cultured colonies for Tet and Lac testing in DH5alpha-Z1</p>
 +
 
 +
<ul>
 +
<li>One colony of pSB1C3-K741002</li>
 +
<li>One colony of pSB1C3-BBa_I13521</li>
 +
<li>In LB+Cm</li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 201: Line 277:
<a id="jul5"><h2> July 5 </h2></a>
<a id="jul5"><h2> July 5 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: Insert crRNAs into pdCas9 backbone</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Saved stocks of 12 strains in 15% glycerol at -80C</span></p><ul class="c7 lst-kix_awzwxkxw8dr3-0 start"><li class="c1"><span>4 tubes each of potential GFP1,2,3 crRNA inserts in pdCas9</span></li></ul><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Miniprepped 12 tubes of pdCas9 with potential GFP-1,2,3 inserts</span></p><ul class="c7 lst-kix_wuplxnpgyfk7-0 start"><li class="c1"><span>4 tubes each </span></li><li class="c1"><span>Eluted in 50 uL EB, all concentrations 100-200 ng/uL</span></li></ul>
+
<p class="obj">Objective: Insert crRNAs into pdCas9 backbone</p>
 +
<p>Saved stocks of 12 strains in 15% glycerol at -80C</p>
-
<p class="obj"><span class="c3">Objective: Test effectiveness of DH5alpha-Z1 strain</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Saved stock of pSB1C3-K741002 in DH5alpha (15% glycerol at -80C)</span></p><ul class="c7 lst-kix_qmh244fhqrkd-0 start"><li class="c1"><span>Culture of I13521 did not grow</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Cultured another colony of pSB1C3-BBa_I13521</span></p><ul class="c7 lst-kix_z207qv64fwzp-0 start"><li class="c1"><span>from 7/3 plate</span></li></ul>
+
<ul>
 +
<li>4 tubes each of potential GFP1,2,3 crRNA inserts in pdCas9</li>
 +
</ul>
 +
<p>Miniprepped 12 tubes of pdCas9 with potential GFP-1,2,3 inserts</p>
 +
 
 +
<ul>
 +
<li>4 tubes each </li>
 +
<li>Eluted in 50 uL EB, all concentrations 100-200 ng/uL</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</p>
 +
<p>Saved stock of pSB1C3-K741002 in DH5alpha (15% glycerol at -80C)</p>
 +
 
 +
<ul>
 +
<li>Culture of I13521 did not grow</li>
 +
</ul>
 +
<p>Cultured another colony of pSB1C3-BBa_I13521</p>
 +
 
 +
<ul>
 +
<li>from 7/3 plate</li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 210: Line 307:
<a id="jul6"><h2> July 6 </h2></a>
<a id="jul6"><h2> July 6 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: Test effectiveness of DH5alpha-Z1 strain</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Saved stock of pSB1C3-I13521 in DH5alpha (15% glycerol at -80C)</span></p>
+
<p class="obj">Objective: Test effectiveness of DH5alpha-Z1 strain</p>
 +
<p>Saved stock of pSB1C3-I13521 in DH5alpha (15% glycerol at -80C)</p>
</div>
</div>
</div>
</div>
Line 217: Line 315:
<a id="jul7"><h2> July 7 </h2></a>
<a id="jul7"><h2> July 7 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: insert crRNAs GFP1,2,3 into pdCas9</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Analytical restriction digest </span></p><ul class="c7 lst-kix_of6w1kl76c4d-0 start"><li class="c1"><span>12 tubes with potential inserts (4 per crRNA)</span></li><li class="c1"><span>pdCas9 as a control</span></li><li class="c1"><span>BsaI and XbaI in cutsmart for 1.5 hrs</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Agarose gel of restriction digest</span></p><ul class="c7 lst-kix_naowbi8l9hai-0 start"><li class="c1"><span>Gel 1:</span></li></ul><ul class="c7 lst-kix_naowbi8l9hai-1 start"><li class="c2"><span>1-4: pdCas9-crRNA-GFP1</span></li><li class="c2"><span>5-8: pdCas9-crRNA-GFP2</span></li><li class="c2"><span>9-12: pdCas9-crRNA-GFP3</span></li>
+
<p class="obj">Objective: insert crRNAs GFP1,2,3 into pdCas9</p>
 +
<p>Analytical restriction digest </p>
 +
 
 +
<ul>
 +
<li>12 tubes with potential inserts (4 per crRNA)</li>
 +
<li>pdCas9 as a control</li>
 +
<li>BsaI and XbaI in cutsmart for 1.5 hrs</li>
 +
</ul>
 +
<p>Agarose gel of restriction digest</p>
 +
 
 +
<ul>
 +
<li>Gel 1:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>1-4: pdCas9-crRNA-GFP1</li>
 +
<li>5-8: pdCas9-crRNA-GFP2</li>
 +
<li>9-12: pdCas9-crRNA-GFP3</li>
<!--https://2014.igem.org/File:GEL_7-6-14_AJC.png or https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png-->
<!--https://2014.igem.org/File:GEL_7-6-14_AJC.png or https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png-->
<li><a href="https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png"></a></li>
<li><a href="https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/d4/GEL_7-6-14_AJC.png"></a></li>
-
</ul><ul class="c7 lst-kix_naowbi8l9hai-0"><li class="c1"><span>Gel 2:</span></li></ul><ul class="c7 lst-kix_naowbi8l9hai-1 start"><li class="c2"><span>pdCas9 (control)</span></li>
+
</ul>
 +
 
 +
<ul>
 +
<li>Gel 2:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pdCas9 (control)</li>
<!--https://2014.igem.org/File:GEL_7-6-14_AJC_2.png or https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png-->
<!--https://2014.igem.org/File:GEL_7-6-14_AJC_2.png or https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png-->
<li><a href="https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png"></a></li>
<li><a href="https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/4e/GEL_7-6-14_AJC_2.png"></a></li>
-
</ul><ul class="c7 lst-kix_naowbi8l9hai-0"><li class="c1"><span>Expected single band ~9.3 kb if BsaI cut and ligation worked</span></li><li class="c1"><span>Control and uncut failures expect two bands, 5.0 and 4.2 kb</span></li><li class="c1"><span class="c4">Results: all 12 samples have single band</span></li></ul><ul class="c7 lst-kix_naowbi8l9hai-1 start"><li class="c2"><span>No uncut plasmids</span></li><li class="c2"><span>Could still be recircularized with no insert</span></li></ul><p class="c6"><span>gel pictures:</span></p><p class="c6 c12"><span></span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Anneal oligos of 3 crRNA-GFP inserts</span></p><ul class="c7 lst-kix_6bzdop9p7sjl-0 start"><li class="c1"><span>Following Dewran&rsquo;s crRNA protocol</span></li><li class="c1"><span>2 uL each oligo diluted in PBS to 20 uL</span></li><li class="c1"><span>Placed in 98C water bath, cooled gradually to rt</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Phosphorylate oligo inserts of crRNAs</span></p><ul class="c7 lst-kix_6bzdop9p7sjl-0"><li class="c1"><span>5 uL annealed oligo mix in 20uL 1x ligase buffer with 1uL PNK</span></li><li class="c1"><span>30 mins at 37C, then deactivated for 20 mins at 65C</span></li><li class="c1"><span>Diluted 1/50</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Ligation </span></p><ul class="c7 lst-kix_6sksc9cavkjf-0 start"><li class="c1"><span>75 ng backbone = 0.6 uL Repeat scaffold or 2.5 uL pdCas9</span></li></ul><ul class="c7 lst-kix_6sksc9cavkjf-1 start"><li class="c2"><span>Both BsaI cut and CIP treated 7/3/14</span></li></ul><ul class="c7 lst-kix_6sksc9cavkjf-0"><li class="c1"><span>1.5 uL diluted, phosphorylated, annealed oligo insert</span></li><li class="c1"><span>6 experimental tubes + 2 BO controls and 3 IO controls</span></li><li class="c1"><span>Transformed into iGEM CCEC (DH5alpha) and plated on LB+Cm</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Cultured 2 colonies from pdCas9 plate</span></p><ul class="c7 lst-kix_b41cwnif73y0-0 start"><li class="c1"><span>For Charlie to try his hands at ligation</span></li></ul>
+
</ul>
-
<p class="obj"><span class="c3">Objective: Prepare various backbones for multiple-plasmid use</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Transformed two distribution kit parts</span></p><ul class="c7 lst-kix_9n9njswl4wfq-0 start"><li class="c1"><span>Plate 4-6H: pSB4K5-J04450</span></li></ul><ul class="c7 lst-kix_9n9njswl4wfq-1 start"><li class="c2"><span>backbone contains pSC101 origin with Kan resistance</span></li></ul><ul class="c7 lst-kix_9n9njswl4wfq-0"><li class="c1"><span>Plate 4-18A: pSB3K3-I20260</span></li></ul><ul class="c7 lst-kix_9n9njswl4wfq-1 start"><li class="c2"><span>backbone contains p15A origin with Kan resistance</span></li><li class="c2"><span>On LB+G418 plates </span></li></ul><p class="c6 c16"><span class="c4">Streaked plate from frozen stock</span></p><ul class="c7 lst-kix_gaiki19jvu68-0 start"><li class="c1"><span>pSB6A1-J04450</span></li></ul><ul class="c7 lst-kix_gaiki19jvu68-1 start"><li class="c2"><span>backbone contains pMB1 origin with Amp resistance</span></li><li class="c2"><span>on LB+Amp</span></li></ul>
+
<ul>
 +
<li>Expected single band ~9.3 kb if BsaI cut and ligation worked</li>
 +
<li>Control and uncut failures expect two bands, 5.0 and 4.2 kb</li>
 +
<li>Results: all 12 samples have single band</li>
 +
</ul>
-
<p class="obj"><span class="c3">Objective: Test pTet and pLac in DH5alphaZ1 strain</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Streaked plates from frozen stock</span></p><ul class="c7 lst-kix_6nicclcjpyxr-0 start"><li class="c1"><span>pSB1C3-K741002 in DH5alpha-Z1</span></li></ul><ul class="c7 lst-kix_6nicclcjpyxr-1 start"><li class="c2"><span>contains pLac-GFP</span></li></ul><ul class="c7 lst-kix_6nicclcjpyxr-0"><li class="c1"><span>pSB1C3-I13521 in DH5alpha-ZI</span></li></ul><ul class="c7 lst-kix_6nicclcjpyxr-1 start"><li class="c2"><span>contains pTet-RFP</span></li></ul><ul class="c7 lst-kix_6nicclcjpyxr-0"><li class="c1"><span>On LB+Cm plates</span></li></ul>
+
<ul>
 +
<li>No uncut plasmids</li>
 +
<li>Could still be recircularized with no insert</li>
 +
</ul>
 +
<p>gel pictures:</p>
 +
 
 +
<p>Anneal oligos of 3 crRNA-GFP inserts</p>
 +
 
 +
<ul>
 +
<li>Following Dewran&rsquo;s crRNA protocol</li>
 +
<li>2 uL each oligo diluted in PBS to 20 uL</li>
 +
<li>Placed in 98C water bath, cooled gradually to rt</li>
 +
</ul>
 +
<p>Phosphorylate oligo inserts of crRNAs</p>
 +
 
 +
<ul>
 +
<li>5 uL annealed oligo mix in 20uL 1x ligase buffer with 1uL PNK</li>
 +
<li>30 mins at 37C, then deactivated for 20 mins at 65C</li>
 +
<li>Diluted 1/50</li>
 +
</ul>
 +
<p>Ligation </p>
 +
 
 +
<ul>
 +
<li>75 ng backbone = 0.6 uL Repeat scaffold or 2.5 uL pdCas9</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Both BsaI cut and CIP treated 7/3/14</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>1.5 uL diluted, phosphorylated, annealed oligo insert</li>
 +
<li>6 experimental tubes + 2 BO controls and 3 IO controls</li>
 +
<li>Transformed into iGEM CCEC (DH5alpha) and plated on LB+Cm</li>
 +
</ul>
 +
<p>Cultured 2 colonies from pdCas9 plate</p>
 +
 
 +
<ul>
 +
<li>For Charlie to try his hands at ligation</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Prepare various backbones for multiple-plasmid use</p>
 +
<p>Transformed two distribution kit parts</p>
 +
 
 +
<ul>
 +
<li>Plate 4-6H: pSB4K5-J04450</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>backbone contains pSC101 origin with Kan resistance</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Plate 4-18A: pSB3K3-I20260</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>backbone contains p15A origin with Kan resistance</li>
 +
<li>On LB+G418 plates </li>
 +
</ul>
 +
<p>Streaked plate from frozen stock</p>
 +
 
 +
<ul>
 +
<li>pSB6A1-J04450</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>backbone contains pMB1 origin with Amp resistance</li>
 +
<li>on LB+Amp</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Test pTet and pLac in DH5alphaZ1 strain</p>
 +
<p>Streaked plates from frozen stock</p>
 +
 
 +
<ul>
 +
<li>pSB1C3-K741002 in DH5alpha-Z1</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>contains pLac-GFP</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pSB1C3-I13521 in DH5alpha-ZI</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>contains pTet-RFP</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>On LB+Cm plates</li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 236: Line 454:
<a id="jul8"><h2> July 8 </h2></a>
<a id="jul8"><h2> July 8 </h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj"><span class="c3">Objective: insert crRNAs into pdCas9</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Plate results from 7/7 transformation</span></p><ul class="c7 lst-kix_76nbwrx263o-0 start"><li class="c1"><span>pdCas9-crRNAs: ~50 colonies each</span></li><li class="c1"><span>pdCas9 BO: 100s of colonies</span></li><li class="c1"><span>Repeat-crRNAs: 100s of colonies</span></li><li class="c1"><span>Repeat BO: ~1000 colonies</span></li><li class="c1"><span>IO controls: 0 colonies, ~15 for crRNA-GFP3</span></li><li class="c1"><span>Notes: unsure why BO controls have more colonies than experimental</span></li></ul><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Restriction digest of 12 samples from 7/5 prep + pdCas9 control</span></p><ul class="c7 lst-kix_fw20mkn20m6q-0 start"><li class="c1"><span>With SacI/NheI</span></li></ul><ul class="c7 lst-kix_fw20mkn20m6q-1 start"><li class="c2"><span>Should show difference between insert and recircularized backbone</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Made and ran 1.5% agarose gel in TBE</span></p><ul class="c7 lst-kix_px57lbouyv0k-0 start"><li class="c1"><span>for better resolution of small band differences in pdCas9 with/without insert</span></li></ul><ul class="c7 lst-kix_px57lbouyv0k-1 start"><li class="c2"><span>1: pdCas9 (control)</span></li><li class="c2"><span>2-5: pdCas9-crRNA-GFP1 (1-4)</span></li><li class="c2"><span>6-9: pdCas9-crRNA-GFP2 (1-4)</span></li><li class="c2"><span>10-13: pdCas9-crRNA-GFP3 (1-4)</span></li><li class="c2"><span>14: pdCas9 (control)</span></li></ul><ul class="c7 lst-kix_px57lbouyv0k-0"><li class="c1"><span>expected: </span></li></ul><ul class="c7 lst-kix_px57lbouyv0k-1 start"><li class="c2"><span>3.1, 2.8, and 2.7 kb bands</span></li><li class="c2"><span>585 bp band with no insert, 620 bp band with insert (pdCas9 620bp)</span></li></ul><ul class="c7 lst-kix_px57lbouyv0k-0"><li class="c1"><span class="c4">Results: One sample, crRNA-GFP3-3, has larger band matching control</span></li></ul><ul class="c7 lst-kix_px57lbouyv0k-1 start"><li class="c2"><span>This colony may be correct, should be sequenced</span></li><li class="c2"><span>Others have smaller band, are probably incorrect</span></li></ul><p class="c6 c16"><span class="c4">Culture 40 colonies from existing plates to screen for crRNA inserts</span></p><ul class="c7 lst-kix_twnnvl56136k-0 start"><li class="c1"><span>10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/3/14</span></li><li class="c1"><span>10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/7/14</span></li><li class="c1"><span>in LB+Cm</span></li></ul>
+
<p class="obj">Objective: insert crRNAs into pdCas9</p>
 +
<p>Plate results from 7/7 transformation</p>
-
<p class="obj"><span class="c3">Objective: Prepare various backbones for multiple-plasmid use</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Prepared LB+Kan medium</span></p><ul class="c7 lst-kix_wtdfzi8k039s-0 start"><li class="c1"><span>50 ug/mL final concentration Kanamycin</span></li></ul><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Cultured colonies from transformations and stock streaking</span></p><ul class="c7 lst-kix_6064784bi77i-0 start"><li class="c1"><span>3 colonies each from 3 plasmids:</span></li></ul><ul class="c7 lst-kix_6064784bi77i-1 start"><li class="c2"><span>pSB6A1-J04450 in LB+Amp</span></li><li class="c2"><span>pSB3K3-I20260 in LB+Kan</span></li><li class="c2"><span>pSB4K5-J04450 in LB+Kan</span></li></ul>
+
<ul>
 +
<li>pdCas9-crRNAs: ~50 colonies each</li>
 +
<li>pdCas9 BO: 100s of colonies</li>
 +
<li>Repeat-crRNAs: 100s of colonies</li>
 +
<li>Repeat BO: ~1000 colonies</li>
 +
<li>IO controls: 0 colonies, ~15 for crRNA-GFP3</li>
 +
<li>Notes: unsure why BO controls have more colonies than experimental</li>
 +
</ul>
 +
<p>Restriction digest of 12 samples from 7/5 prep + pdCas9 control</p>
-
<p class="obj"><span class="c3">Objective: Test pLac and pTet in DH5alpha-Z1</span></p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span>Notes:</span></p><ul class="c7 lst-kix_k0c06f1k8dpr-0 start"><li class="c1"><span>Need to obtain aTc for pTet testing</span></li></ul><ul class="c7 lst-kix_k0c06f1k8dpr-1 start"><li class="c2"><span>Tried to use Doxycycline, but had trouble with solubility in ethanol (?)</span></li></ul><ul class="c7 lst-kix_k0c06f1k8dpr-0"><li class="c1"><span>pLac is not the final promoter to be used, a different variant will be used</span></li></ul><ul class="c7 lst-kix_k0c06f1k8dpr-1 start"><li class="c2"><span>with no glucose dependency</span></li></ul><ul class="c7 lst-kix_k0c06f1k8dpr-0"><li class="c1"><span>Flow testing of pLac and pTet was suspended until these changes are made</span></li></ul>
+
<ul>
 +
<li>With SacI/NheI</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Should show difference between insert and recircularized backbone</li>
 +
</ul>
 +
<p>Made and ran 1.5% agarose gel in TBE</p>
 +
 
 +
<ul>
 +
<li>for better resolution of small band differences in pdCas9 with/without insert</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>1: pdCas9 (control)</li>
 +
<li>2-5: pdCas9-crRNA-GFP1 (1-4)</li>
 +
<li>6-9: pdCas9-crRNA-GFP2 (1-4)</li>
 +
<li>10-13: pdCas9-crRNA-GFP3 (1-4)</li>
 +
<li>14: pdCas9 (control)</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>expected: </li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>3.1, 2.8, and 2.7 kb bands</li>
 +
<li>585 bp band with no insert, 620 bp band with insert (pdCas9 620bp)</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Results: One sample, crRNA-GFP3-3, has larger band matching control</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>This colony may be correct, should be sequenced</li>
 +
<li>Others have smaller band, are probably incorrect</li>
 +
</ul>
 +
<p>Culture 40 colonies from existing plates to screen for crRNA inserts</p>
 +
 
 +
<ul>
 +
<li>10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/3/14</li>
 +
<li>10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/7/14</li>
 +
<li>in LB+Cm</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Prepare various backbones for multiple-plasmid use</p>
 +
<p>Prepared LB+Kan medium</p>
 +
 
 +
<ul>
 +
<li>50 ug/mL final concentration Kanamycin</li>
 +
</ul>
 +
<p>Cultured colonies from transformations and stock streaking</p>
 +
 
 +
<ul>
 +
<li>3 colonies each from 3 plasmids:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pSB6A1-J04450 in LB+Amp</li>
 +
<li>pSB3K3-I20260 in LB+Kan</li>
 +
<li>pSB4K5-J04450 in LB+Kan</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Test pLac and pTet in DH5alpha-Z1</p>
 +
<p>Notes:</p>
 +
 
 +
<ul>
 +
<li>Need to obtain aTc for pTet testing</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Tried to use Doxycycline, but had trouble with solubility in ethanol (?)</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pLac is not the final promoter to be used, a different variant will be used</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>with no glucose dependency</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Flow testing of pLac and pTet was suspended until these changes are made</li>
 +
</ul>
</div>
</div>
</div>
</div>
 +
<div class="day">
<div class="day">
Line 920: Line 1,233:
</div>
</div>
</div>
</div>
 +
 +
 +
 +
<div class="day">
<div class="day">
Line 927: Line 1,244:
<div class="lab">
<div class="lab">
<p>Minipreps</p>
<p>Minipreps</p>
-
<ul><li>4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation</li></ul>
+
<ul>
-
<ul><li>4 copies of pSB1C3-R0011-I13500 from 7/15 ligation</li></ul>
+
<li>4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation</li>
 +
</ul>
 +
<ul>
 +
<li>4 copies of pSB1C3-R0011-I13500 from 7/15 ligation</li>
 +
</ul>
<p>Analytical digest of minipreps</p>
<p>Analytical digest of minipreps</p>
-
<ul class="c7 lst-kix_lxs7wstahkp3-0 start"><li class="c1"><span class="c0">pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI</span></li><li class="c1"><span class="c0">pSB1C3-R0011-I13500 with MfeI/NdeI/SacI</span></li></ul>
+
<ul>
 +
<li>pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI</li>
 +
<li>pSB1C3-R0011-I13500 with MfeI/NdeI/SacI</li>
 +
</ul>
<p>Agarose gel of digest results:</p>
<p>Agarose gel of digest results:</p>
-
<ul class="c7 lst-kix_vio5o0mh9rst-0 start"><li class="c1"><span class="c0">Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, &lt;0.1 kb bands)</span></li><li class="c1"><span class="c0">Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)</span></li><li class="c1"><span class="c0 c4">Results: </span></li></ul><ul class="c7 lst-kix_vio5o0mh9rst-1 start"><li class="c2"><span class="c0 c4">R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly</span></li><li class="c2"><span class="c0 c4">R0011-I13500 does not look correct (only one band at 2.0 kb)</span></li></ul>
+
<ul>
 +
<li>Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, &lt;0.1 kb bands)</li>
 +
<li>Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)</li>
 +
<li>Results: </li>
 +
</ul>
 +
<ul>
 +
 
 +
<li>R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly</li>
 +
<li>R0011-I13500 does not look correct (only one band at 2.0 kb)</li>
 +
</ul>
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
<div class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</div>
-
<p>Colony PCR on pSB1C3-dCas9-tracrRNA plate</p><ul class="c7 lst-kix_3muxqzmhmgsp-0 start"><li class="c1"><span class="c0">Ligation plate from 7/15/14</span></li><li class="c1"><span class="c0">15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC</span></li><li class="c1"><span class="c0">oligos SB1C3-up1 and SB1C3-dn1</span></li><li class="c1"><span class="c0">annealed at 65C, extension 2 mins</span></li></ul>
+
<p>Colony PCR on pSB1C3-dCas9-tracrRNA plate</p>
 +
<ul>
 +
<li>Ligation plate from 7/15/14</li>
 +
<li>15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC</li>
 +
<li>oligos SB1C3-up1 and SB1C3-dn1</li>
 +
<li>annealed at 65C, extension 2 mins</li>
 +
</ul>
-
<p>Agarose gel of colony PCR</span></p><ul class="c7 lst-kix_nxjmmijl9oy9-0 start"><li class="c1"><span class="c0 c4">results: primer-bands only (no successes)</span></li></ul>
+
<p>Agarose gel of colony PCR</p>
 +
<ul>
 +
<li>results: primer-bands only (no successes)</li>
 +
</ul>
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
<div class="obj">Objective: Switch K608012 into pSB6A1</div>
-
<p>Culture colonies</p><ul class="c7 lst-kix_6jc4x55ftesi-0 start"><li class="c1"><span class="c0">2 copies of pSB6A1-J04450 (from 6-9-14 plate)</span></li><li class="c1"><span class="c0">2 copies of pSB1C3-K608012</span>
+
<p>Culture colonies</p>
 +
<ul>
 +
<li>2 copies of pSB6A1-J04450 (from 6-9-14 plate)</li>
 +
<li>2 copies of pSB1C3-K608012
</p>
</p>
</div>
</div>
</div>
</div>
 +
<div class="day">
<div class="day">
Line 950: Line 1,296:
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">PCR of dCas9-tracrRNA from pdCas9</span></p><ul class="c7 lst-kix_tj97n1d5w3ra-0 start"><li class="c1"><span class="c0">8 tubes, each with 1 uL pdCas9 template at 1 ng/uL</span></li><li class="c1"><span class="c0">oligos dCas9tracr-up and dCas9-tracr-dn</span></li><li class="c1"><span class="c0">2 min extension, 64C anneal</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of PCR product</span></p><ul class="c7 lst-kix_y2bntayn8fir-0 start"><li class="c1"><span class="c0">All 8 tubes look successful (band ~4 kb)</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">PCR cleanup of dCas9-tracrRNA</span></p><ul class="c7 lst-kix_bnq2w6ulrpf3-0 start"><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">Into 2 tubes, 30 uL each</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Prep-scale digest of PCR product</span></p><ul class="c7 lst-kix_i477fpkrgmqo-0 start"><li class="c1"><span class="c0">with PstI/XbaI/DpnI</span></li><li class="c1"><span class="c0">in 100 uL each for 3 hrs at 37C</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Treat pSB1C3 digest with CIP</span></p><ul class="c7 lst-kix_nqsd62y9j32h-0 start"><li class="c1"><span class="c0">1 hr at 37C</span></li></ul>
+
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
 +
<p>PCR of dCas9-tracrRNA from pdCas9</p>
 +
<ul>
 +
<li>8 tubes, each with 1 uL pdCas9 template at 1 ng/uL</li>
 +
<li>oligos dCas9tracr-up and dCas9-tracr-dn</li>
 +
<li>2 min extension, 64C anneal</li>
 +
</ul>
 +
<p>Agarose gel of PCR product</p>
 +
<ul>
 +
<li>All 8 tubes look successful (band ~4 kb)</li>
 +
</ul>
 +
<p>PCR cleanup of dCas9-tracrRNA</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>Into 2 tubes, 30 uL each</li>
 +
</ul>
 +
<p>Prep-scale digest of PCR product</p>
 +
<ul>
 +
<li>with PstI/XbaI/DpnI</li>
 +
<li>in 100 uL each for 3 hrs at 37C</li>
 +
</ul>
 +
<p>Treat pSB1C3 digest with CIP</p>
 +
<ul>
 +
<li>1 hr at 37C</li>
 +
</ul>
-
<p class="obj">Objective: Switch K608012 into pSB6A1</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Miniprep pSB1C3-K608012 and pSB6A1-J04450</span></p><ul class="c7 lst-kix_1un0c8hdx4by-0 start"><li class="c1"><span class="c0">1 copy each (the second copy of each did not grow in culture)</span></li><li class="c1"><span class="c0">concentrations:</span></li></ul><ul class="c7 lst-kix_1un0c8hdx4by-1 start"><li class="c2"><span class="c0">pSB1C3-K608012: 344 ng/uL</span></li><li class="c2"><span class="c0">pSB6A1-J04450: 421.5 ng/uL</span></li></ul><p class="c6 c16"><span class="c0 c4">Prep-scale digest of K608012 and pSB6A1</span></p><ul class="c7 lst-kix_2ge12mwdaeuo-0 start"><li class="c1"><span class="c0">50 uL DNA in 100 uL reaction</span></li><li class="c1"><span class="c0">Both with EcoRI and PstI</span></li><li class="c1"><span class="c0">3 hrs at 37C</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">CIP treatment of pSB6A1 and heat-inactivation</span></p><ul class="c7 lst-kix_eemrs6rebxix-0 start"><li class="c1"><span class="c0">2.5 uL CIP added to digest tube</span></li><li class="c1"><span class="c0">Both digests returned to 37C for 1 hr</span></li><li class="c1"><span class="c0">Then 30 mins at 80C to heat-inactivate</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Gel extraction of pSB6A1 and K608012</span></p><ul class="c7 lst-kix_vx9o6fdwru2r-0 start"><li class="c1"><span class="c0">Gel had no EtBr added, and was discarded (product was lost)</span></li></ul>
+
<p class="obj">Objective: Switch K608012 into pSB6A1</p>
 +
<p>Miniprep pSB1C3-K608012 and pSB6A1-J04450</p>
 +
<ul>
 +
<li>1 copy each (the second copy of each did not grow in culture)</li>
 +
<li>concentrations:</li>
 +
</ul>
 +
<ul>
 +
<li>pSB1C3-K608012: 344 ng/uL</li>
 +
<li>pSB6A1-J04450: 421.5 ng/uL</li>
 +
</ul>
 +
<p>Prep-scale digest of K608012 and pSB6A1</p>
 +
<ul>
 +
<li>50 uL DNA in 100 uL reaction</li>
 +
<li>Both with EcoRI and PstI</li>
 +
<li>3 hrs at 37C</li>
 +
</ul>
 +
<p>CIP treatment of pSB6A1 and heat-inactivation</p>
 +
<ul>
 +
<li>2.5 uL CIP added to digest tube</li>
 +
<li>Both digests returned to 37C for 1 hr</li>
 +
<li>Then 30 mins at 80C to heat-inactivate</li>
 +
</ul>
 +
<p>Gel extraction of pSB6A1 and K608012</p>
 +
<ul>
 +
<li>Gel had no EtBr added, and was discarded (product was lost)</li>
 +
</ul>
</div>
</div>
</div>
</div>
 +
Line 961: Line 1,357:
<a id="jul20"><h2>July 20</h2></a>
<a id="jul20"><h2>July 20</h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: culture colonies for various purposes</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Cultured colonies</span></p><ul class="c7 lst-kix_i3248osi0yoh-0 start"><li class="c1"><span class="c0">4 copies pSB6A1-J04450</span></li></ul><ul class="c7 lst-kix_i3248osi0yoh-1 start"><li class="c2"><span class="c0">2 from older plate, 2 from newer</span></li></ul><ul class="c7 lst-kix_i3248osi0yoh-0"><li class="c1"><span class="c0">4 copies pSB1C3-K608012</span></li></ul><ul class="c7 lst-kix_i3248osi0yoh-1 start"><li class="c2"><span class="c0">2 from older plate, 2 from newer</span></li></ul><ul class="c7 lst-kix_i3248osi0yoh-0"><li class="c1"><span class="c0">4 copies pSB1C3-B0030</span></li></ul><ul class="c7 lst-kix_i3248osi0yoh-1 start"><li class="c2"><span class="c0">To obtain pSB1C3 without gel extraction</span></li></ul><ul class="c7 lst-kix_i3248osi0yoh-0"><li class="c1"><span class="c0">4 copies pSB1C3-R0011</span></li><li class="c1"><span class="c0">4 copies pSB1C3-I13500</span></li></ul>
+
<p class="obj">Objective: culture colonies for various purposes</p>
 +
<p>Cultured colonies</p>
 +
<ul>
 +
<li>4 copies pSB6A1-J04450</li>
 +
</ul>
 +
<ul>
 +
<li>2 from older plate, 2 from newer</li>
 +
</ul>
 +
<ul>
 +
<li>4 copies pSB1C3-K608012</li>
 +
</ul>
 +
<ul>
 +
<li>2 from older plate, 2 from newer</li>
 +
</ul>
 +
<ul>
 +
<li>4 copies pSB1C3-B0030</li>
 +
</ul>
 +
<ul>
 +
<li>To obtain pSB1C3 without gel extraction</li>
 +
</ul>
 +
<ul>
 +
<li>4 copies pSB1C3-R0011</li>
 +
<li>4 copies pSB1C3-I13500</li>
 +
</ul>
</div>
</div>
</div>
</div>
 +
<div class="day">
<div class="day">
<a id="jul21"><h2>July 21</h2></a>
<a id="jul21"><h2>July 21</h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: Sequence to confirm constructs</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Sequencing reaction and Order #1634</span></p><ul class="c7 lst-kix_m1yhwi5p15u2-0 start"><li class="c1"><span class="c0">1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1</span></li><li class="c1"><span class="c0">5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1</span></li><li class="c1"><span class="c0">9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1</span></li><li class="c1"><span class="c0">11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1</span></li><li class="c1"><span class="c0">Big Dye, buffer version 1.1</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Order 1634 Results:</span></p><ul class="c7 lst-kix_9x4q31caynac-0 start"><li class="c1"><span class="c0">pdCas9-GFP3 copies 3, 6, and 8 are correct</span></li></ul><ul class="c7 lst-kix_9x4q31caynac-1 start"><li class="c2"><span class="c0">copy 5 has a single mutation</span></li></ul><ul class="c7 lst-kix_9x4q31caynac-0"><li class="c1"><span class="c0">anti-tracrRNA: both copies are correct</span></li></ul><ul class="c7 lst-kix_9x4q31caynac-1 start"><li class="c2"><span class="c0">copy 1 was a cleaner, more reliable </span></li></ul>
+
<p class="obj">Objective: Sequence to confirm constructs</p>
 +
<p>Sequencing reaction and Order #1634</p>
 +
<ul>
 +
<li>1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1</li>
 +
<li>5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1</li>
 +
<li>9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1</li>
 +
<li>11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1</li>
 +
<li>Big Dye, buffer version 1.1</li>
 +
</ul>
 +
<p>Order 1634 Results:</p>
 +
<ul>
 +
<li>pdCas9-GFP3 copies 3, 6, and 8 are correct</li>
 +
</ul>
 +
<ul>
 +
<li>copy 5 has a single mutation</li>
 +
</ul>
 +
<ul>
 +
<li>anti-tracrRNA: both copies are correct</li>
 +
</ul>
 +
<ul>
 +
<li>copy 1 was a cleaner, more reliable </li>
 +
</ul>
-
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Minipreps</span></p><ul class="c7 lst-kix_72bobqm6ajha-0 start"><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">4 tubes of pSB1C3-R0011</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-1 start"><li class="c2"><span class="c0">concentrations 255.7, 169.2, 204.8, 151.2 ng/uL</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-0"><li class="c1"><span class="c0">4 tubes of pSB1C3-I13500</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-1 start"><li class="c2"><span class="c0">concentrations 148.9, 214.2, 260.7, 432.5</span></li></ul><p class="c6 c16"><span class="c0 c4">Prep scale digests of pSB1C3-R0011 and I13500</span></p><ul class="c7 lst-kix_vi5muatdut0f-0 start"><li class="c1"><span class="c0">pSB1C3-R0011 with SpeI/PstI, samples 1,3</span></li><li class="c1"><span class="c0">pSB1C3-I13500 with XbaI/PstI, samples 1,3</span></li><li class="c1"><span class="c0">50 uL DNA in 100 uL each reaction, 2 tubes per construct</span></li><li class="c1"><span class="c0">3 hrs at 37C</span></li><li class="c1"><span class="c0">Added CIP (2.5 uL) to pSB1C3-R0011 reaction</span></li><li class="c1"><span class="c0">1 more hr for both at 37C</span></li><li class="c1"><span class="c0">Heat inactivation of both reactions: 30 mins at 80C</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Gel extraction of I13500</span></p><ul class="c7 lst-kix_n4hwg2vuko5n-0 start"><li class="c1"><span class="c0">extraction gel 1, lanes 1 and 2</span></li><li class="c1"><span class="c0">Zymoclean prep kit</span></li><li class="c1"><span class="c0">sample 1 (lane 1): 300 mg gel yields</span></li><li class="c1"><span class="c0">sample 3 (lane 2): 550 mg gel yields</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">PCR cleanup of pSB1C3-R0011 </span></p><ul class="c7 lst-kix_m384ate7isqf-0 start"><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">Two tubes of digest combined into one tube product</span></li><li class="c1"><span class="c0">Concentration </span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Ligation of pSB1C3-R0011 and I13500</span></p><ul class="c7 lst-kix_htvbtc6p5aj8-0 start"><li class="c1"><span class="c0">Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL</span></li><li class="c1"><span class="c0">New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL</span></li><li class="c1"><span class="c0">Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL</span></li><li class="c1"><span class="c0">New insert (I13500 extracted 7/21): 300 ng = 3 uL</span></li><li class="c1"><span class="c0">Ligations numbered 1-8:</span></li></ul><ul class="c7 lst-kix_htvbtc6p5aj8-1 start"><li class="c2"><span class="c0">1. OB+OI</span></li><li class="c2"><span class="c0">2. OB+NI</span></li><li class="c2"><span class="c0">3. NB+OI</span></li><li class="c2"><span class="c0">4. NB+NI</span></li><li class="c2"><span class="c0">5. OB control</span></li><li class="c2"><span class="c0">6. OI control</span></li><li class="c2"><span class="c0">7. NB control</span></li><li class="c2"><span class="c0">8. NI control</span></li></ul><ul class="c7 lst-kix_htvbtc6p5aj8-0"><li class="c1"><span class="c0">1 hr at rt</span></li><li class="c1"><span class="c0">Transformed via heat shock into DH5alphas, plated on LB+Cm</span></li></ul>
+
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
 +
<p>Minipreps</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>4 tubes of pSB1C3-R0011</li>
 +
</ul>
 +
<ul>
 +
<li>concentrations 255.7, 169.2, 204.8, 151.2 ng/uL</li>
 +
</ul>
 +
<ul>
 +
<li>4 tubes of pSB1C3-I13500</li>
 +
</ul>
 +
<ul>
 +
<li>concentrations 148.9, 214.2, 260.7, 432.5</li>
 +
</ul>
 +
<p>Prep scale digests of pSB1C3-R0011 and I13500</p>
 +
<ul>
-
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Minipreps</span></p><ul class="c7 lst-kix_72bobqm6ajha-0"><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">4 tubes of pSB1C3-B0030</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-1 start"><li class="c2"><span class="c0">to obtain pSB1C3 without gel extraction</span></li><li class="c2"><span class="c0">concentrations 116.8, 99.9, 115.3, 145.1 ng/uL</span></li></ul><p class="c6 c16"><span class="c0 c4">PCR cleanup of dCas9-tracrRNA PCR</span></p><ul class="c7 lst-kix_cqt91kerquvr-0 start"><li class="c1"><span class="c0">Product digested 7/18 with XbaI/PstI/DpnI</span></li><li class="c1"><span class="c0">Concentration 80.5 ng/uL in 30 uL</span></li><li class="c1"><span class="c0">Heat inactivation of cleaned product, 30 mins at 80C</span></li></ul><p class="c6 c16"><span class="c0 c4">Prep scale digest of pSB1C3-B0030</span></p><ul class="c7 lst-kix_vi5muatdut0f-0"><li class="c1"><span class="c0">With XbaI/PstI, samples 1,3</span></li><li class="c1"><span class="c0">50 uL DNA in 100 uL each reaction, 2 tubes </span></li><li class="c1"><span class="c0">3 hrs at 37C</span></li><li class="c1"><span class="c0">Added CIP (2.5 uL) </span></li><li class="c1"><span class="c0">1 more hr for both at 37C</span></li><li class="c1"><span class="c0">Heat inactivation: 30 mins at 80C</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">PCR cleanup of pSB1C3 </span></p><ul class="c7 lst-kix_m384ate7isqf-0"><li class="c1"><span class="c0">B0030 should not be retained</span></li><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">Two tubes of digest combined into one tube product</span></li><li class="c1"><span class="c0">Concentration </span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Ligation of pSB1C3 and dCas9-tracrRNA</span></p><ul class="c7 lst-kix_htvbtc6p5aj8-0"><li class="c1"><span class="c0">Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL</span></li><li class="c1"><span class="c0">Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL</span></li><li class="c1"><span class="c0">New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL</span></li><li class="c1"><span class="c0">Ligations numbered 9-13:</span></li></ul><ul class="c7 lst-kix_htvbtc6p5aj8-1 start"><li class="c2"><span class="c0">9. B+OI</span></li><li class="c2"><span class="c0">10. B+NI</span></li><li class="c2"><span class="c0">11. B control</span></li><li class="c2"><span class="c0">12. OI control</span></li><li class="c2"><span class="c0">13. NI control</span></li></ul><ul class="c7 lst-kix_htvbtc6p5aj8-0"><li class="c1"><span class="c0">1 hr at rt</span></li><li class="c1"><span class="c0">Transformed via heat shock into DH5alphas, plated on LB+Cm</span></li></ul>
+
<li>pSB1C3-R0011 with SpeI/PstI, samples 1,3</li>
 +
<li>pSB1C3-I13500 with XbaI/PstI, samples 1,3</li>
 +
<li>50 uL DNA in 100 uL each reaction, 2 tubes per construct</li>
 +
<li>3 hrs at 37C</li>
 +
<li>Added CIP (2.5 uL) to pSB1C3-R0011 reaction</li>
 +
<li>1 more hr for both at 37C</li>
 +
<li>Heat inactivation of both reactions: 30 mins at 80C</li>
 +
</ul>
 +
<p>Gel extraction of I13500</p>
 +
<ul>
 +
<li>extraction gel 1, lanes 1 and 2</li>
 +
<li>Zymoclean prep kit</li>
 +
<li>sample 1 (lane 1): 300 mg gel yields</li>
 +
<li>sample 3 (lane 2): 550 mg gel yields</li>
 +
</ul>
 +
<p>PCR cleanup of pSB1C3-R0011 </p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>Two tubes of digest combined into one tube product</li>
 +
<li>Concentration </li>
 +
</ul>
 +
<p>Ligation of pSB1C3-R0011 and I13500</p>
 +
<ul>
 +
<li>Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL</li>
 +
<li>New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL</li>
 +
<li>Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL</li>
 +
<li>New insert (I13500 extracted 7/21): 300 ng = 3 uL</li>
 +
<li>Ligations numbered 1-8:</li>
 +
</ul>
 +
<ul>
 +
<li>1. OB+OI</li>
 +
<li>2. OB+NI</li>
 +
<li>3. NB+OI</li>
 +
<li>4. NB+NI</li>
 +
<li>5. OB control</li>
 +
<li>6. OI control</li>
 +
<li>7. NB control</li>
 +
<li>8. NI control</li>
 +
</ul>
 +
<ul>
 +
<li>1 hr at rt</li>
 +
<li>Transformed via heat shock into DH5alphas, plated on LB+Cm</li>
 +
</ul>
-
<p class="obj">Objective: Switch K608012 into pSB6A1 </span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Minipreps</span></p><ul class="c7 lst-kix_72bobqm6ajha-0"><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">4 tubes of pSB6A1-J04450</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-1 start"><li class="c2"><span class="c0">concentrations 189.3, 432.5, 688.7, 487.3 ng/uL</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-0"><li class="c1"><span class="c0">4 tubes of pSB1C3-K608012</span></li></ul><ul class="c7 lst-kix_72bobqm6ajha-1 start"><li class="c2"><span class="c0">concentrations 68.8, 91.6, 346.1, ng/uL</span></li></ul><p class="c6 c16"><span class="c0 c4">Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012</span></p><ul class="c7 lst-kix_vi5muatdut0f-0"><li class="c1"><span class="c0">pSB6A1-J04450 with XbaI/SpeI, samples 1,3</span></li><li class="c1"><span class="c0">pSB1C3-K608012 with EcoRI/PstI, samples 1,3</span></li><li class="c1"><span class="c0">50 uL DNA in 100 uL each reaction, 2 tubes per construct</span></li><li class="c1"><span class="c0">3 hrs at 37C</span></li><li class="c1"><span class="c0">Added CIP (2.5 uL) to pSB6A1-J04450 reaction</span></li><li class="c1"><span class="c0">1 more hr for both at 37C</span></li><li class="c1"><span class="c0">Heat inactivation of both reactions: 30 mins at 80C</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Gel extraction of pSB6A1 and K608012</span></p><ul class="c7 lst-kix_n4hwg2vuko5n-0"><li class="c1"><span class="c0">K608012: gel 1, lane 3 and gel 2, lane 1</span></li><li class="c1"><span class="c0">pSB6A1: gel 2, lanes 2 and 3</span></li><li class="c1"><span class="c0">Zymoclean prep kit</span></li><li class="c1"><span class="c0">K608012 sample 1 (lane 1-3): 300 mg gel yields</span></li><li class="c1"><span class="c0">K608012 sample 3 (lane 2-1): 550 mg gel yields</span></li><li class="c1"><span class="c0">pSB6A1 sample 1 (lane 2-2):</span></li><li class="c1"><span class="c0">pSB6A1 sample 3 (lane 2-3):</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Ligation of pSB6A1 and K608012</span></p><ul class="c7 lst-kix_htvbtc6p5aj8-0"><li class="c1"><span class="c0">Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL</span></li><li class="c1"><span class="c0">Old insert (K608012 extracted 7/15): 300 ng = 4 uL</span></li><li class="c1"><span class="c0">Ligations numbered 14-16:</span></li></ul><ul class="c7 lst-kix_htvbtc6p5aj8-1 start"><li class="c2"><span class="c0">14. B+I</span></li><li class="c2"><span class="c0">15. B control</span></li><li class="c2"><span class="c0">16. I control</span></li></ul><ul class="c7 lst-kix_htvbtc6p5aj8-0"><li class="c1"><span class="c0">1 hr at rt</span></li><li class="c1"><span class="c0">Transformed via heat shock into DH5alphas, plated on LB+Amp</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Gibson of pSB6A1 and K608012</span></p><ul class="c7 lst-kix_iu15755crc4w-0 start"><li class="c1"><span class="c0">New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL</span></li><li class="c1"><span class="c0">New insert (K608012 extracted 7/21): 300 ng = 1.7 uL</span></li><li class="c1"><span class="c0">Numbered 17-19:</span></li></ul><ul class="c7 lst-kix_iu15755crc4w-1 start"><li class="c2"><span class="c0">17. B+I</span></li><li class="c2"><span class="c0">18. B control</span></li><li class="c2"><span class="c0">19. I control</span></li></ul><ul class="c7 lst-kix_iu15755crc4w-0"><li class="c1"><span class="c0">15 uL Mert&rsquo;s DIY Gibson mix</span></li><li class="c1"><span class="c0">1 hr at 60C</span></li><li class="c1"><span class="c0">Transformed via heat shock into DH5alphas, plated on LB+Amp</span></li></ul><p class="c6 c23"><span class="c0 c4">SLIC of pSB6A1 and K608012</span></p><ul class="c7 lst-kix_iu15755crc4w-0"><li class="c1"><span class="c0">New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL</span></li><li class="c1"><span class="c0">New insert (K608012 extracted 7/21): 300 ng = 1.7 uL</span></li><li class="c1"><span class="c0">Numbered 20-22:</span></li></ul><ul class="c7 lst-kix_iu15755crc4w-1 start"><li class="c2"><span class="c0">20. B+I</span></li><li class="c2"><span class="c0">21. B control</span></li><li class="c2"><span class="c0">22. I control</span></li></ul><ul class="c7 lst-kix_iu15755crc4w-0"><li class="c1"><span class="c0">2.5 mins at rt, then 10 mins at 4C</span></li><li class="c1"><span class="c0">Transformed via heat shock into DH5alphas, plated on LB+Amp</span></li></ul>
+
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
 +
<p>Minipreps</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>4 tubes of pSB1C3-B0030</li>
 +
</ul>
 +
<ul>
 +
<li>to obtain pSB1C3 without gel extraction</li>
 +
<li>concentrations 116.8, 99.9, 115.3, 145.1 ng/uL</li>
 +
</ul>
 +
<p>PCR cleanup of dCas9-tracrRNA PCR</p>
 +
<ul>
 +
<li>Product digested 7/18 with XbaI/PstI/DpnI</li>
 +
<li>Concentration 80.5 ng/uL in 30 uL</li>
 +
<li>Heat inactivation of cleaned product, 30 mins at 80C</li>
 +
</ul>
 +
<p>Prep scale digest of pSB1C3-B0030</p>
 +
<ul>
 +
<li>With XbaI/PstI, samples 1,3</li>
 +
<li>50 uL DNA in 100 uL each reaction, 2 tubes </li>
 +
<li>3 hrs at 37C</li>
 +
<li>Added CIP (2.5 uL) </li>
 +
<li>1 more hr for both at 37C</li>
 +
<li>Heat inactivation: 30 mins at 80C</li>
 +
</ul>
 +
<p>PCR cleanup of pSB1C3 </p>
 +
<ul>
 +
<li>B0030 should not be retained</li>
 +
<li>Qiagen kit</li>
 +
<li>Two tubes of digest combined into one tube product</li>
 +
<li>Concentration </li>
 +
</ul>
 +
<p>Ligation of pSB1C3 and dCas9-tracrRNA</p>
 +
<ul>
 +
<li>Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL</li>
 +
<li>Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL</li>
 +
<li>New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL</li>
 +
<li>Ligations numbered 9-13:</li>
 +
</ul>
 +
<ul>
 +
<li>9. B+OI</li>
 +
<li>10. B+NI</li>
 +
<li>11. B control</li>
 +
<li>12. OI control</li>
 +
<li>13. NI control</li>
 +
</ul>
 +
<ul>
 +
<li>1 hr at rt</li>
 +
<li>Transformed via heat shock into DH5alphas, plated on LB+Cm</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Switch K608012 into pSB6A1 </p>
 +
<p>Minipreps</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>4 tubes of pSB6A1-J04450</li>
 +
</ul>
 +
<ul>
 +
<li>concentrations 189.3, 432.5, 688.7, 487.3 ng/uL</li>
 +
</ul>
 +
<ul>
 +
<li>4 tubes of pSB1C3-K608012</li>
 +
</ul>
 +
<ul>
 +
<li>concentrations 68.8, 91.6, 346.1, ng/uL</li>
 +
</ul>
 +
<p>Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012</p>
 +
<ul>
 +
<li>pSB6A1-J04450 with XbaI/SpeI, samples 1,3</li>
 +
<li>pSB1C3-K608012 with EcoRI/PstI, samples 1,3</li>
 +
<li>50 uL DNA in 100 uL each reaction, 2 tubes per construct</li>
 +
<li>3 hrs at 37C</li>
 +
<li>Added CIP (2.5 uL) to pSB6A1-J04450 reaction</li>
 +
<li>1 more hr for both at 37C</li>
 +
<li>Heat inactivation of both reactions: 30 mins at 80C</li>
 +
</ul>
 +
<p>Gel extraction of pSB6A1 and K608012</p>
 +
<ul>
 +
<li>K608012: gel 1, lane 3 and gel 2, lane 1</li>
 +
<li>pSB6A1: gel 2, lanes 2 and 3</li>
 +
<li>Zymoclean prep kit</li>
 +
<li>K608012 sample 1 (lane 1-3): 300 mg gel yields</li>
 +
<li>K608012 sample 3 (lane 2-1): 550 mg gel yields</li>
 +
<li>pSB6A1 sample 1 (lane 2-2):</li>
 +
<li>pSB6A1 sample 3 (lane 2-3):</li>
 +
</ul>
 +
<p>Ligation of pSB6A1 and K608012</p>
 +
<ul>
 +
<li>Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL</li>
 +
<li>Old insert (K608012 extracted 7/15): 300 ng = 4 uL</li>
 +
<li>Ligations numbered 14-16:</li>
 +
</ul>
 +
<ul>
 +
<li>14. B+I</li>
 +
<li>15. B control</li>
 +
<li>16. I control</li>
 +
</ul>
 +
<ul>
 +
<li>1 hr at rt</li>
 +
<li>Transformed via heat shock into DH5alphas, plated on LB+Amp</li>
 +
</ul>
 +
<p>Gibson of pSB6A1 and K608012</p>
 +
<ul>
 +
<li>New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL</li>
 +
<li>New insert (K608012 extracted 7/21): 300 ng = 1.7 uL</li>
 +
<li>Numbered 17-19:</li>
 +
</ul>
 +
<ul>
 +
<li>17. B+I</li>
 +
<li>18. B control</li>
 +
<li>19. I control</li>
 +
</ul>
 +
<ul>
 +
<li>15 uL Mert&rsquo;s DIY Gibson mix</li>
 +
<li>1 hr at 60C</li>
 +
<li>Transformed via heat shock into DH5alphas, plated on LB+Amp</li>
 +
</ul>
 +
<p>SLIC of pSB6A1 and K608012</p>
 +
<ul>
 +
<li>New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL</li>
 +
<li>New insert (K608012 extracted 7/21): 300 ng = 1.7 uL</li>
 +
<li>Numbered 20-22:</li>
 +
</ul>
 +
<ul>
 +
<li>20. B+I</li>
 +
<li>21. B control</li>
 +
<li>22. I control</li>
 +
</ul>
 +
<ul>
 +
<li>2.5 mins at rt, then 10 mins at 4C</li>
 +
<li>Transformed via heat shock into DH5alphas, plated on LB+Amp</li>
 +
</ul>
</div>
</div>
</div>
</div>
 +
<div class="day">
<div class="day">
<a id="jul22"><h2>July 22</h2></a>
<a id="jul22"><h2>July 22</h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: Create pSB1C3-R0011-I13500</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Plate results:</span></p><ul class="c7 lst-kix_za8cwgv9ejy9-0 start"><li class="c1"><span class="c0">All four experimental plates had lots of colonies</span></li><li class="c1"><span class="c0">both Backbone-only controls had similar colony numbers to experimentals</span></li><li class="c1"><span class="c0">Insert-only controls had no colonies</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Colony PCR</span></p><ul class="c7 lst-kix_x93cj9yoq980-0 start"><li class="c1"><span class="c0">4 copies each of 4 experimental plates = 16 colonies</span></li><li class="c1"><span class="c0">Oligos SB1C3-up and SB1C3-dn with Taq polymerase</span></li><li class="c1"><span class="c0">66C anneal temp, 2 min extension time</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of colony PCR results</span></p><ul class="c7 lst-kix_fhwuvigf7aza-0 start"><li class="c1"><span class="c0">1-4: OB+OI pSB1C3-R0011-I13500</span></li><li class="c1"><span class="c0">5-8: OB+NI pSB1C3-R0011-I13500</span></li><li class="c1"><span class="c0">9-12: NB+OI pSB1C3-R0011-I13500</span></li><li class="c1"><span class="c0">13-16: NB+NI pSB1C3-R0011-I13500</span></li><li class="c1"><span class="c0">17-20: B+OI pSB1C3-dCas9-tracrRNA</span></li><li class="c1"><span class="c0">21-24: B+NI pSB1C3-dCas9-tracrRNA</span></li></ul><ul class="c7 lst-kix_x93cj9yoq980-0"><li class="c1"><span class="c0 c4">Results: no successful bands on any colonies</span></li></ul><ul class="c7 lst-kix_x93cj9yoq980-1 start"><li class="c2"><span class="c0">Just primer-dimers or small amplified bands?</span></li></ul>
+
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
 +
<p>Plate results:</p>
 +
<ul>
 +
<li>All four experimental plates had lots of colonies</li>
 +
<li>both Backbone-only controls had similar colony numbers to experimentals</li>
 +
<li>Insert-only controls had no colonies</li>
 +
</ul>
 +
<p>Colony PCR</p>
 +
<ul>
 +
<li>4 copies each of 4 experimental plates = 16 colonies</li>
 +
<li>Oligos SB1C3-up and SB1C3-dn with Taq polymerase</li>
 +
<li>66C anneal temp, 2 min extension time</li>
 +
</ul>
 +
<p>Agarose gel of colony PCR results</p>
 +
<ul>
 +
<li>1-4: OB+OI pSB1C3-R0011-I13500</li>
 +
<li>5-8: OB+NI pSB1C3-R0011-I13500</li>
 +
<li>9-12: NB+OI pSB1C3-R0011-I13500</li>
 +
<li>13-16: NB+NI pSB1C3-R0011-I13500</li>
 +
<li>17-20: B+OI pSB1C3-dCas9-tracrRNA</li>
 +
<li>21-24: B+NI pSB1C3-dCas9-tracrRNA</li>
 +
</ul>
 +
<ul>
 +
<li>Results: no successful bands on any colonies</li>
 +
</ul>
 +
<ul>
 +
<li>Just primer-dimers or small amplified bands?</li>
 +
</ul>
-
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</span></p><p class="c6 c23"><span class="c0 c4">Plate results:</span></p><ul class="c7 lst-kix_b6dewkecyn7r-0 start"><li class="c1"><span class="c0">Both experimental plates had lots of colonies</span></li><li class="c1"><span class="c0">Backbone-only controls had similar colony numbers to experimentals</span></li><li class="c1"><span class="c0">Insert-only controls had no colonies</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Colony PCR</span></p><ul class="c7 lst-kix_6cggtci34gds-0 start"><li class="c1"><span class="c0">4 copies each of 2 experimental plates = 8 colonies</span></li><li class="c1"><span class="c0">Oligos SB1C3-up and SB1C3-dn with Taq polymerase</span></li><li class="c1"><span class="c0">66C anneal temp, 2 min extension time</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of colony PCR results</span></p><ul class="c7 lst-kix_y5z1tmgxiu3z-0 start"><li class="c1"><span class="c0">see above for gel order</span></li></ul><ul class="c7 lst-kix_6cggtci34gds-0"><li class="c1"><span class="c0 c4">Results: no successful bands on any colonies</span></li></ul><ul class="c7 lst-kix_6cggtci34gds-1 start"><li class="c2"><span class="c0">Just primer-dimers or small amplified bands?</span></li></ul>
+
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
 +
<p>Plate results:</p>
 +
<ul>
 +
<li>Both experimental plates had lots of colonies</li>
 +
<li>Backbone-only controls had similar colony numbers to experimentals</li>
 +
<li>Insert-only controls had no colonies</li>
 +
</ul>
 +
<p>Colony PCR</p>
 +
<ul>
 +
<li>4 copies each of 2 experimental plates = 8 colonies</li>
 +
<li>Oligos SB1C3-up and SB1C3-dn with Taq polymerase</li>
 +
<li>66C anneal temp, 2 min extension time</li>
 +
</ul>
 +
<p>Agarose gel of colony PCR results</p>
 +
<ul>
 +
<li>see above for gel order</li>
 +
</ul>
 +
<ul>
 +
<li>Results: no successful bands on any colonies</li>
 +
</ul>
 +
<ul>
 +
<li>Just primer-dimers or small amplified bands?</li>
 +
</ul>
-
<p class="obj">Objective: Switch K608012 into pSB6A1</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Plate results:</span></p><ul class="c7 lst-kix_ymlx12jxx0i-0 start"><li class="c1"><span class="c0">No colonies on Gibson or SLIC plates</span></li><li class="c1"><span class="c0">3 colonies on Ligation plate</span></li><li class="c1"><span class="c0">A few colonies on controls, but not many</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Cultured colonies</span></p><ul class="c7 lst-kix_qc8gdvrh3rv9-0 start"><li class="c1"><span class="c0">3 colonies of potential pSB6A1-K608012 from Ligation plate</span></li><li class="c1"><span class="c0">in LB+Amp</span></li></ul><p class="c6 c23"><span class="c0 c4">Ligation of K608012 and pSB6A1</span></p><ul class="c7 lst-kix_awrmfaud01gy-0 start"><li class="c1"><span class="c0">Same constructs as 7/21 ligation</span></li><li class="c1"><span class="c0">This time let recover in SOC for 2 hrs instead of 1 hr</span></li><li class="c1"><span class="c0">Plated on LB+Amp</span></li></ul>
+
<p class="obj">Objective: Switch K608012 into pSB6A1</p>
 +
<p>Plate results:</p>
 +
<ul>
 +
<li>No colonies on Gibson or SLIC plates</li>
 +
<li>3 colonies on Ligation plate</li>
 +
<li>A few colonies on controls, but not many</li>
 +
</ul>
 +
<p>Cultured colonies</p>
 +
<ul>
 +
<li>3 colonies of potential pSB6A1-K608012 from Ligation plate</li>
 +
<li>in LB+Amp</li>
 +
</ul>
 +
<p>Ligation of K608012 and pSB6A1</p>
 +
<ul>
 +
<li>Same constructs as 7/21 ligation</li>
 +
<li>This time let recover in SOC for 2 hrs instead of 1 hr</li>
 +
<li>Plated on LB+Amp</li>
 +
</ul>
-
<p class="obj">Objective: Try putting K608012 into pSB1A2 instead</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Transformation from kit plate</span></p><ul class="c7 lst-kix_luzoqg4o02e1-0 start"><li class="c1"><span class="c0">Plate 4-1N</span></li></ul><ul class="c7 lst-kix_luzoqg4o02e1-1 start"><li class="c2"><span class="c0">pSB1A2-B0034</span></li></ul><ul class="c7 lst-kix_luzoqg4o02e1-0"><li class="c1"><span class="c0">Plated on LB+Amp</span></li></ul>
+
<p class="obj">Objective: Try putting K608012 into pSB1A2 instead</p>
 +
<p>Transformation from kit plate</p>
 +
<ul>
 +
<li>Plate 4-1N</li>
 +
</ul>
 +
<ul>
 +
<li>pSB1A2-B0034</li>
 +
</ul>
 +
<ul>
 +
<li>Plated on LB+Amp</li>
 +
</ul>
</div>
</div>
</div>
</div>
 +
<div class="day">
<div class="day">
<a id="jul23"><h2>July 23</h2></a>
<a id="jul23"><h2>July 23</h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: Switch K608012 into pSB6A1</span></p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Miniprep 3 copies of potential pSB6A1-K608012</span></p><ul class="c7 lst-kix_zdt8wvfie4o6-0 start"><li class="c1"><span class="c0">Qiagen kit</span></li><li class="c1"><span class="c0">Saved stocks in 15% glycerol</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Analytical digest of minipreps</span></p><ul class="c7 lst-kix_ux221p3uqppm-0 start"><li class="c1"><span class="c0">With NdeI in Cutsmart, 10 uL final volume</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of analytical digest</span></p><ul class="c7 lst-kix_3phjo1jb9910-0 start"><li class="c1"><span class="c0 c4">Results:</span></li></ul><ul class="c7 lst-kix_3phjo1jb9910-1 start"><li class="c2"><span class="c0">Copies 1 and 2 are strange and incorrect</span></li><li class="c2"><span class="c0">Copy 3 looks correct</span></li><li class="c2"><span class="c0">Need to check with more digests</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Analytical digest to check promising sample</span></p><ul class="c7 lst-kix_6xwnoxjdyuoe-0 start"><li class="c1"><span class="c0">Copy 3 in 3 different reactions</span></li></ul><ul class="c7 lst-kix_6xwnoxjdyuoe-1 start"><li class="c2"><span class="c0">EcoRI/SpeI</span></li><li class="c2"><span class="c0">EcoRI/MfeI</span></li><li class="c2"><span class="c0">NcoI/PvuI</span></li></ul><ul class="c7 lst-kix_6xwnoxjdyuoe-0"><li class="c1"><span class="c0">Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI</span></li><li class="c1"><span class="c0">Control: pSB1C3-K608012 with MfeI/NcoI</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of analytical digest</span></p><ul class="c7 lst-kix_asllv2qk19ks-0 start"><li class="c1"><span class="c0">1. Ligation 3 with EcoRI/SpeI</span></li><li class="c1"><span class="c0">2. Ligation 3 with EcoRI/MfeI</span></li><li class="c1"><span class="c0">3. Ligation 3 with NcoI/PvuI</span></li><li class="c1"><span class="c0">4. pSB6A1-J04450 with EcoRI/SpeI/PvuI</span></li><li class="c1"><span class="c0">5. pSB1C3-K608012 with MfeI/NcoI</span></li></ul><ul class="c7 lst-kix_9bqui6dplzq-0 start"><li class="c1"><span class="c0 c4">Results:</span></li></ul><ul class="c7 lst-kix_9bqui6dplzq-1 start"><li class="c2"><span class="c0 c4">Sample looks good</span></li><li class="c2"><span class="c0">Incorrect samples 1 and 2 glycerol stocks thrown out</span></li></ul>
+
<p class="obj">Objective: Switch K608012 into pSB6A1</p>
 +
<p>Miniprep 3 copies of potential pSB6A1-K608012</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>Saved stocks in 15% glycerol</li>
 +
</ul>
 +
<p>Analytical digest of minipreps</p>
 +
<ul>
 +
<li>With NdeI in Cutsmart, 10 uL final volume</li>
 +
</ul>
 +
<p>Agarose gel of analytical digest</p>
 +
<ul>
 +
<li>Results:</li>
 +
</ul>
 +
<ul>
 +
<li>Copies 1 and 2 are strange and incorrect</li>
 +
<li>Copy 3 looks correct</li>
 +
<li>Need to check with more digests</li>
 +
</ul>
 +
<p>Analytical digest to check promising sample</p>
 +
<ul>
 +
<li>Copy 3 in 3 different reactions</li>
 +
</ul>
 +
<ul>
 +
<li>EcoRI/SpeI</li>
 +
<li>EcoRI/MfeI</li>
 +
<li>NcoI/PvuI</li>
 +
</ul>
 +
<ul>
 +
<li>Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI</li>
 +
<li>Control: pSB1C3-K608012 with MfeI/NcoI</li>
 +
</ul>
 +
<p>Agarose gel of analytical digest</p>
 +
<ul>
 +
<li>1. Ligation 3 with EcoRI/SpeI</li>
 +
<li>2. Ligation 3 with EcoRI/MfeI</li>
 +
<li>3. Ligation 3 with NcoI/PvuI</li>
 +
<li>4. pSB6A1-J04450 with EcoRI/SpeI/PvuI</li>
 +
<li>5. pSB1C3-K608012 with MfeI/NcoI</li>
 +
</ul>
 +
<ul>
 +
<li>Results:</li>
 +
</ul>
 +
<ul>
 +
<li>Sample looks good</li>
 +
<li>Incorrect samples 1 and 2 glycerol stocks thrown out</li>
 +
</ul>
-
<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Treat pSB4K5 with CIP</span></p><ul class="c7 lst-kix_u6g5u0d0gudn-0 start"><li class="c1"><span class="c0">1.25 uL CIP in 25 uL reaction</span></li><li class="c1"><span class="c0">pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Prep-scale digest to obtain R0040-anti-tracrRNA</span></p><ul class="c7 lst-kix_xanwattg67ve-0 start"><li class="c1"><span class="c0">pSB1C3-R0040-anti-tracrRNA </span></li><li class="c1"><span class="c0">with EcoRI/SpeI</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Gel extraction and cleanup of R0040-anti-tracrRNA</span></p><ul class="c7 lst-kix_lkyl03f9go16-0 start"><li class="c1"><span class="c0">Extracted smaller band</span></li><li class="c1"><span class="c0">Attempted to clean up using Qiagen prep kit</span></li></ul><ul class="c7 lst-kix_lkyl03f9go16-1 start"><li class="c2"><span class="c0">dissolved with Zymoclean ADB</span></li><li class="c2"><span class="c0">Then followed Qiagen PCR cleanup protocol</span></li><li class="c2"><span class="c0">510 mg gel yielded little or no DNA </span></li></ul>
+
<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p>
 +
<p>Treat pSB4K5 with CIP</p>
 +
<ul>
 +
<li>1.25 uL CIP in 25 uL reaction</li>
 +
<li>pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9</li>
 +
</ul>
 +
<p>Prep-scale digest to obtain R0040-anti-tracrRNA</p>
 +
<ul>
 +
<li>pSB1C3-R0040-anti-tracrRNA </li>
 +
<li>with EcoRI/SpeI</li>
 +
</ul>
 +
<p>Gel extraction and cleanup of R0040-anti-tracrRNA</p>
 +
<ul>
 +
<li>Extracted smaller band</li>
 +
<li>Attempted to clean up using Qiagen prep kit</li>
 +
</ul>
 +
<ul>
 +
<li>dissolved with Zymoclean ADB</li>
 +
<li>Then followed Qiagen PCR cleanup protocol</li>
 +
<li>510 mg gel yielded little or no DNA </li>
 +
</ul>
-
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Colony PCR</span></p><ul class="c7 lst-kix_3zx0kxwyyebn-0 start"><li class="c1"><span class="c0">8 colonies from 7/21 ligation plates</span></li><li class="c1"><span class="c0">Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Agarose gel of colony PCR results</span></p><ul class="c7 lst-kix_tv00453xv5n-0 start"><li class="c1"><span class="c0">No successes</span></li><li class="c1"><span class="c0">Controls did not work properly either</span></li><li class="c1"><span class="c0">colony PCR does not seem to be effective--need to just pick colonies</span></li></ul><p class="c6"><span class="c0">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Culture colonies</span></p><ul class="c7 lst-kix_8cfdswkyiqqc-0 start"><li class="c1"><span class="c0">8 colonies of potential pSB1C3-R0011-I13500</span></li><li class="c1"><span class="c0">From 7/21 ligation plates (colonies from today&rsquo;s colony PCR)</span></li></ul>
+
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
 +
<p>Colony PCR</p>
 +
<ul>
 +
<li>8 colonies from 7/21 ligation plates</li>
 +
<li>Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011</li>
 +
</ul>
 +
<p>Agarose gel of colony PCR results</p>
 +
<ul>
 +
<li>No successes</li>
 +
<li>Controls did not work properly either</li>
 +
<li>colony PCR does not seem to be effective--need to just pick colonies</li>
 +
</ul>
 +
<p>Culture colonies</p>
 +
<ul>
 +
<li>8 colonies of potential pSB1C3-R0011-I13500</li>
 +
<li>From 7/21 ligation plates (colonies from today&rsquo;s colony PCR)</li>
 +
</ul>
-
<p class="obj">Objective: Measurement interlab study</p><p class="c6"><span class="c0 c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c0 c4">Transformations from kit plates</span></p><ul class="c7 lst-kix_wt60ybrwcvom-0 start"><li class="c1"><span class="c0">Plate 1-20K</span></li></ul><ul class="c7 lst-kix_wt60ybrwcvom-1 start"><li class="c2"><span class="c0">pSB1C3-K823005</span></li></ul><ul class="c7 lst-kix_wt60ybrwcvom-0"><li class="c1"><span class="c0">Plate 1-22I</span></li></ul><ul class="c7 lst-kix_wt60ybrwcvom-1 start"><li class="c2"><span class="c0">pSB1C3-K823012</span></li></ul><ul class="c7 lst-kix_wt60ybrwcvom-0"><li class="c1"><span class="c0">Plate 2-24B</span></li></ul><ul class="c7 lst-kix_wt60ybrwcvom-1 start"><li class="c2"><span class="c0">pSB1C3-E0240</span></li></ul>
+
<p class="obj">Objective: Measurement interlab study</p>
 +
<p>Transformations from kit plates</p>
 +
<ul>
 +
<li>Plate 1-20K</li>
 +
</ul>
 +
<ul>
 +
<li>pSB1C3-K823005</li>
 +
</ul>
 +
<ul>
 +
<li>Plate 1-22I</li>
 +
</ul>
 +
<ul>
 +
<li>pSB1C3-K823012</li>
 +
</ul>
 +
<ul>
 +
<li>Plate 2-24B</li>
 +
</ul>
 +
<ul>
 +
<li>pSB1C3-E0240</li>
 +
</ul>
</div>
</div>
</div>
</div>
Line 1,368: Line 2,177:
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
<p>Analytical digest of minipreps</p>
<p>Analytical digest of minipreps</p>
-
<ul>class="c7 lst-kix_kxcu5bd6bzm0-0 start">
+
<ul>
<li>Colonies 5 and 14 of potential pSB1C3-R0011-I13500</li>
<li>Colonies 5 and 14 of potential pSB1C3-R0011-I13500</li>
<li>with MfeI</li>
<li>with MfeI</li>
Line 1,601: Line 2,410:
<a id="jul29"><h2>July 29</h2></a>
<a id="jul29"><h2>July 29</h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Minipreps</span></p><ul class="c7 lst-kix_el6oy7wmrj12-0 start"><li class="c1"><span>pSB4K5-J04450 (4 copies)</span></li><li class="c1"><span>pSB1C3-R0040-anti-tracrRNA (4 copies)</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Prep-scale digest</span></p><ul class="c7 lst-kix_12tm50mrytxp-0 start"><li class="c1"><span>pSB4K5-J04450 (4 copies) with EcoRI/SpeI</span></li><li class="c1"><span>pSB1C3-R0040-anti-tracrRNA (4 copies) with EcoRI/SpeI</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Gel extraction and cleanup of digests</span></p><ul class="c7 lst-kix_nq5huiafibyu-0 start"><li class="c1"><span>Cut pSB4K5 backbone from 4 copies of pSB4K5-J04450</span></li></ul><ul class="c7 lst-kix_nq5huiafibyu-1 start"><li class="c2"><span>2 digests per gel well</span></li><li class="c2"><span>2.04 g total gel eluted into two tubes:</span></li></ul><ul class="c7 lst-kix_nq5huiafibyu-2 start"><li class="c5"><span>51.4 ng/uL and 94.5 ng/uL, 30 uL each</span></li></ul><ul class="c7 lst-kix_nq5huiafibyu-0"><li class="c1"><span>Cut R0040-anti-tracrRNA insert from pSB1C3-R0040-anti-tracrRNA</span></li></ul><ul class="c7 lst-kix_nq5huiafibyu-1 start"><li class="c2"><span>2 digests per gel well</span></li><li class="c2"><span>1.61 g total gel eluted into two tubes:</span></li></ul><ul class="c7 lst-kix_nq5huiafibyu-2 start"><li class="c5"><span>99.4 ng/uL and 82.3 ng/uL, 30 uL each</span></li></ul>
+
<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5</p>
 +
<p>Minipreps</p>
 +
<ul>
 +
<li>pSB4K5-J04450 (4 copies)</li>
 +
<li>pSB1C3-R0040-anti-tracrRNA (4 copies)</li>
 +
</ul>
-
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Colony PCR</span></p><ul class="c7 lst-kix_k1yrvwrckssi-0 start"><li class="c1"><span>24 colonies of potential pSB1C3-R0011-I13500</span></li></ul><ul class="c7 lst-kix_k1yrvwrckssi-1 start"><li class="c2"><span>8 colonies from 7/15 ligation</span></li><li class="c2"><span>4x4=16 colonies from 4 different 7/21 ligation plates</span></li></ul><ul class="c7 lst-kix_k1yrvwrckssi-0"><li class="c1"><span>Taq polymerase with oligos SB1C3-up and SB1C3-dn</span></li><li class="c1"><span>extension time 45 sec, anneal temp 62C</span></li></ul><p class="c6 c16"><span class="c4">Agarose gel of colony PCR results</span></p><ul class="c7 lst-kix_hby6wpiy31pk-0 start"><li class="c1"><span>colony #19 is only promising result</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Analytical digest</span></p><ul class="c7 lst-kix_2hw1nznzo566-0 start"><li class="c1"><span>pSB1C3-R0011 copies 2, 4</span></li><li class="c1"><span>pSB1C3-I13500 copies 2, 4</span></li><li class="c1"><span>with MfeI/NcoI</span></li><li class="c1"><span>To test whether enzymes or original parts are the source of ligation difficulties</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Agarose gel of digest results</span></p><ul class="c7 lst-kix_9021hr66yhim-0 start"><li class="c1"><span>Order:</span></li></ul><ul class="c7 lst-kix_9021hr66yhim-1 start"><li class="c2"><span>1. pSB1C3-R0011 #2 cut with MfeI/NcoI (expected 2 bands)</span></li><li class="c2"><span>2. pSB1C3-R0011 #4 cut with MfeI/NcoI (expected 2 bands)</span></li><li class="c2"><span>3. pSB1C3-I13500 #2 cut with MfeI/NcoI (expected 3 bands)</span></li><li class="c2"><span>4. pSB1C3-I13500 #4 cut with MfeI/NcoI (expected 3 bands)</span></li></ul><ul class="c7 lst-kix_9021hr66yhim-0"><li class="c1"><span>Results:</span></li></ul><ul class="c7 lst-kix_9021hr66yhim-1 start"><li class="c2"><span>R0011 both copies only show one band</span></li><li class="c2"><span>I13500 both copies appear correct</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Cultured colonies</span></p><ul class="c7 lst-kix_an4xryr2821s-0 start"><li class="c1"><span>Potential pSB1C3-R0011-I13500 #19 from colony PCR</span></li><li class="c1"><span>pSB1C3-R0011 (3 copies) from original transformation </span></li></ul><ul class="c7 lst-kix_an4xryr2821s-1 start"><li class="c2"><span>suspicion that our R0011 is incorrect--could be just bad copies</span></li></ul>
+
<p>Prep-scale digest</p>
 +
<ul>
 +
<li>pSB4K5-J04450 (4 copies) with EcoRI/SpeI</li>
 +
<li>pSB1C3-R0040-anti-tracrRNA (4 copies) with EcoRI/SpeI</li>
 +
</ul>
-
<p class="obj">Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds</p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Culture colonies</span></p><ul class="c7 lst-kix_8pw7x9wk5dxw-0 start"><li class="c1"><span>pSB1C3-R0040 (4 copies)</span></li></ul>
+
<p>Gel extraction and cleanup of digests</p>
 +
<ul>
 +
<li>Cut pSB4K5 backbone from 4 copies of pSB4K5-J04450</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>2 digests per gel well</li>
 +
<li>2.04 g total gel eluted into two tubes:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>51.4 ng/uL and 94.5 ng/uL, 30 uL each</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Cut R0040-anti-tracrRNA insert from pSB1C3-R0040-anti-tracrRNA</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>2 digests per gel well</li>
 +
<li>1.61 g total gel eluted into two tubes:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>99.4 ng/uL and 82.3 ng/uL, 30 uL each</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
 +
<p>Colony PCR</p>
 +
<ul>
 +
<li>24 colonies of potential pSB1C3-R0011-I13500</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>8 colonies from 7/15 ligation</li><li>4x4=16 colonies from 4 different 7/21 ligation plates</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Taq polymerase with oligos SB1C3-up and SB1C3-dn</li>
 +
<li>extension time 45 sec, anneal temp 62C</li>
 +
</ul>
 +
 
 +
<p>Agarose gel of colony PCR results</p>
 +
<ul>
 +
<li>colony #19 is only promising result</li>
 +
</ul>
 +
 
 +
<p>Analytical digest</p>
 +
<ul>
 +
<li>pSB1C3-R0011 copies 2, 4</li>
 +
<li>pSB1C3-I13500 copies 2, 4</li>
 +
<li>with MfeI/NcoI</li>
 +
<li>To test whether enzymes or original parts are the source of ligation difficulties</li>
 +
</ul>
 +
 
 +
<p>Agarose gel of digest results</p>
 +
<ul>
 +
<li>Order:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>1. pSB1C3-R0011 #2 cut with MfeI/NcoI (expected 2 bands)</li>
 +
<li>2. pSB1C3-R0011 #4 cut with MfeI/NcoI (expected 2 bands)</li>
 +
<li>3. pSB1C3-I13500 #2 cut with MfeI/NcoI (expected 3 bands)</li>
 +
<li>4. pSB1C3-I13500 #4 cut with MfeI/NcoI (expected 3 bands)</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Results:</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>R0011 both copies only show one band</li>
 +
<li>I13500 both copies appear correct</li>
 +
</ul>
 +
 
 +
<p>Cultured colonies</p>
 +
<ul>
 +
<li>Potential pSB1C3-R0011-I13500 #19 from colony PCR</li>
 +
<li>pSB1C3-R0011 (3 copies) from original transformation </li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>suspicion that our R0011 is incorrect--could be just bad copies</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds</p>
 +
<p>Culture colonies</p>
 +
<ul>
 +
<li>pSB1C3-R0040 (4 copies)</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Measurement interlab study</p>
 +
<p>Plate results</p>
 +
<ul>
 +
<li>both experimental and both backbone-only plates all had hundreds of colonies</li>
 +
<li>insert-only controls had no colonies</li>
 +
</ul>
 +
 
 +
<p>Colony PCR</p>
 +
<ul>
 +
<li>8 colonies of potential pSB1C3-K823005-E0240</li>
 +
<li>8 colonies of potential pSB1C3-K823012-E0240</li>
 +
<li>Taq polymerase with oligos SB1C3-up and SB1C3-dn</li>
 +
<li>extension time 45 sec, anneal temp 62C</li>
 +
</ul>
 +
 
 +
<p>Agarose gel of colony PCR results</p>
 +
<ul>
 +
<li>1-8: pSB1C3-K823005-E0240</li>
 +
<li>9-16: pSB1C3-K823012-E0240</li>
 +
<li>Results: no successes</li>
 +
</ul>
-
<p class="obj">Objective: Measurement interlab study</p><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Plate results</span></p><ul class="c7 lst-kix_k5qlo2orpwsw-0 start"><li class="c1"><span>both experimental and both backbone-only plates all had hundreds of colonies</span></li><li class="c1"><span>insert-only controls had no colonies</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Colony PCR</span></p><ul class="c7 lst-kix_42svchupjvta-0 start"><li class="c1"><span>8 colonies of potential pSB1C3-K823005-E0240</span></li><li class="c1"><span>8 colonies of potential pSB1C3-K823012-E0240</span></li><li class="c1"><span>Taq polymerase with oligos SB1C3-up and SB1C3-dn</span></li><li class="c1"><span>extension time 45 sec, anneal temp 62C</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Agarose gel of colony PCR results</span></p><ul class="c7 lst-kix_z95ax6p9uzby-0 start"><li class="c1"><span>1-8: pSB1C3-K823005-E0240</span></li><li class="c1"><span>9-16: pSB1C3-K823012-E0240</span></li><li class="c1"><span>Results: no successes</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></p>
 
</div>
</div>
</div>
</div>
 +
<div class="day">
<div class="day">
<a id="jul30"><h2>July 30</h2></a>
<a id="jul30"><h2>July 30</h2></a>
<div class="lab">
<div class="lab">
-
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Minipreps</span></p><ul class="c7 lst-kix_n8p8ttkxpqyn-0 start"><li class="c1"><span>pSB1C3-R0011-I13500 #19</span></li></ul><ul class="c7 lst-kix_n8p8ttkxpqyn-1 start"><li class="c2"><span>concentration 246.4 ng/uL</span></li></ul><ul class="c7 lst-kix_n8p8ttkxpqyn-0"><li class="c1"><span>pSB1C3-R0011 (3 copies)</span></li></ul><ul class="c7 lst-kix_n8p8ttkxpqyn-1 start"><li class="c2"><span>concentrations 187.6, 178.8, 212.8 ng/uL</span></li></ul><p class="c6 c16"><span class="c4">Analytical digest</span></p><ul class="c7 lst-kix_cl1bfo4b9vf3-0 start"><li class="c1"><span>pSB1C3-R0011-I13500 #19</span></li></ul><ul class="c7 lst-kix_cl1bfo4b9vf3-1 start"><li class="c2"><span>three digests in cutsmart, 10 uL final volume each</span></li></ul><ul class="c7 lst-kix_cl1bfo4b9vf3-2 start"><li class="c5"><span>NcoI</span></li><li class="c5"><span>MfeI</span></li><li class="c5"><span>XbaI/PstI</span></li></ul><ul class="c7 lst-kix_cl1bfo4b9vf3-0"><li class="c1"><span>pSB1C3-R0011 (3 copies)</span></li></ul><ul class="c7 lst-kix_cl1bfo4b9vf3-1 start"><li class="c2"><span>with NcoI/MfeI in cutsmart, 10 uL final volume</span></li></ul><p class="c6 c16"><span class="c4">Agarose gel of digest results</span></p><ul class="c7 lst-kix_5kafbeflcf23-0 start"><li class="c1"><span>1. pSB1C3-R0011-I13500 #19 with NcoI</span></li><li class="c1"><span>2. pSB1C3-R0011-I13500 #19 with MfeI</span></li><li class="c1"><span>3. pSB1C3-R0011-I13500 #19 with EcoRI/PstI</span></li><li class="c1"><span>4. pSB1C3-R0011 #1 with NcoI/MfeI</span></li><li class="c1"><span>5. pSB1C3-R0011 #2 with NcoI/MfeI</span></li><li class="c1"><span>6. pSB1C3-R0011 #3 with NcoI/MfeI</span></li><li class="c1"><span>Results: hard to explain</span></li></ul><ul class="c7 lst-kix_5kafbeflcf23-1 start"><li class="c2"><span>R0011-I13500 not correct</span></li><li class="c2"><span>R0011 all three copies only cut once </span></li></ul>
+
<p class="obj">Objective: Create pSB1C3-R0011-I13500</p>
 +
<p>Minipreps</p>
 +
<ul>
 +
<li>pSB1C3-R0011-I13500 #19</li>
 +
</ul>
-
<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5 </p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Treat pSB4K5 with Antarctic Phosphatase</span></p><ul class="c7 lst-kix_l6aais986ia3-0 start"><li class="c1"><span>2 copies digested on 7/29, combined into one tube with 100 uL final volume</span></li><li class="c1"><span>2 hrs at 37C</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">PCR cleanup of AP-treated pSB4K5</span></p><ul class="c7 lst-kix_q6oeyrvbmije-0 start"><li class="c1"><span>Qiagen kit</span></li><li class="c1"><span>concentration 45.2 ng/uL in 50 uL</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Ligation of pSB4K5 and R0040-anti-tracrRNA</span></p><ul class="c7 lst-kix_aomjjfxwafox-0 start"><li class="c1"><span>2 experimental plates with 3x and 6x molar insert</span></li><li class="c1"><span>pSB4K5 backbone (EcoRI/SpeI/AP)100 ng = 2 uL</span></li><li class="c1"><span>R0040-anti-tracrRNA insert (EcoRI/SpeI) 30 ng = 0.3 uL, or 60 ng = 0.6 uL</span></li><li class="c1"><span>Backbone-only and insert-only controls</span></li><li class="c1"><span>Transformed via heat shock and plated on LB+G418</span></li></ul>
+
<ul>
 +
<li>concentration 246.4 ng/uL</li>
 +
</ul>
-
<p class="obj">Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds</p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Minipreps</span></p><ul class="c7 lst-kix_n8p8ttkxpqyn-0"><li class="c1"><span>pSB1C3-R0040 (4 copies)</span></li></ul><ul class="c7 lst-kix_n8p8ttkxpqyn-1 start"><li class="c2"><span>concentrations 47.4, 80.7, 109.7, 193.5</span></li></ul>
+
<ul>
 +
<li>pSB1C3-R0011 (3 copies)</li>
 +
</ul>
-
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p><p class="c6"><span class="c3">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Treat pSB1C3 with Antarctic Phosphatase</span></p><ul class="c7 lst-kix_l6aais986ia3-0"><li class="c1"><span>cleaned copy from 7/28 with 100 uL final volume</span></li><li class="c1"><span>2 hrs at 37C</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">PCR cleanup of AP-treated pSB1C3</span></p><ul class="c7 lst-kix_q6oeyrvbmije-0"><li class="c1"><span>Qiagen kit</span></li><li class="c1"><span>concentration 104.5 ng/uL in 50 uL</span></li></ul><p class="c6"><span>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c4">Ligation of pSB1C3 and dCas9-tracrRNA</span></p><ul class="c7 lst-kix_aomjjfxwafox-0"><li class="c1"><span>2 experimental plates</span></li></ul><ul class="c7 lst-kix_aomjjfxwafox-1 start"><li class="c2"><span>3x molar dCas9-tracrRNA &ldquo;insert&rdquo;</span></li></ul><ul class="c7 lst-kix_aomjjfxwafox-2 start"><li class="c5"><span>pSB1C3 100 ng = 1 uL</span></li><li class="c5"><span>dCas9-tracrRNA 600 ng = 7.5 uL</span></li></ul><ul class="c7 lst-kix_aomjjfxwafox-1"><li class="c2"><span>3x molar pSB1C3 &ldquo;backbone&rdquo;</span></li></ul><ul class="c7 lst-kix_aomjjfxwafox-2 start"><li class="c5"><span>pSB1C3 150 ng = 1.5 uL</span></li><li class="c5"><span>dCas9-tracrRNA 100 ng = 1.2 uL</span></li></ul><ul class="c7 lst-kix_aomjjfxwafox-0"><li class="c1"><span>Backbone-only and insert-only controls</span></li><li class="c1"><span>Transformed via heat shock and plated on LB+Cm</span></li></ul>
+
<ul>
 +
<li>concentrations 187.6, 178.8, 212.8 ng/uL</li>
 +
</ul>
 +
 
 +
<p>Analytical digest</p>
 +
<ul>
 +
<li>pSB1C3-R0011-I13500 #19</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>three digests in cutsmart, 10 uL final volume each</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>NcoI</li>
 +
<li>MfeI</li>
 +
<li>XbaI/PstI</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pSB1C3-R0011 (3 copies)</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>with NcoI/MfeI in cutsmart, 10 uL final volume</li>
 +
</ul>
 +
 
 +
<p>Agarose gel of digest results</p>
 +
<ul>
 +
<li>1. pSB1C3-R0011-I13500 #19 with NcoI</li>
 +
<li>2. pSB1C3-R0011-I13500 #19 with MfeI</li>
 +
<li>3. pSB1C3-R0011-I13500 #19 with EcoRI/PstI</li>
 +
<li>4. pSB1C3-R0011 #1 with NcoI/MfeI</li>
 +
<li>5. pSB1C3-R0011 #2 with NcoI/MfeI</li>
 +
<li>6. pSB1C3-R0011 #3 with NcoI/MfeI</li>
 +
<li>Results: hard to explain</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>R0011-I13500 not correct</li>
 +
<li>R0011 all three copies only cut once </li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Switch R0040-anti-tracrRNA into pSB4K5 </p>
 +
<p>Treat pSB4K5 with Antarctic Phosphatase</p>
 +
<ul>
 +
<li>2 copies digested on 7/29, combined into one tube with 100 uL final volume</li>
 +
<li>2 hrs at 37C</li>
 +
</ul>
 +
 
 +
<p>PCR cleanup of AP-treated pSB4K5</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>concentration 45.2 ng/uL in 50 uL</li>
 +
</ul>
 +
 
 +
<p>Ligation of pSB4K5 and R0040-anti-tracrRNA</p>
 +
<ul>
 +
<li>2 experimental plates with 3x and 6x molar insert</li>
 +
<li>pSB4K5 backbone (EcoRI/SpeI/AP)100 ng = 2 uL</li>
 +
<li>R0040-anti-tracrRNA insert (EcoRI/SpeI) 30 ng = 0.3 uL, or 60 ng = 0.6 uL</li>
 +
<li>Backbone-only and insert-only controls</li><li>Transformed via heat shock and plated on LB+G418</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds</p>
 +
<p>Minipreps</p>
 +
<ul>
 +
<li>pSB1C3-R0040 (4 copies)</li>
 +
</ul>
 +
<ul>
 +
<li>concentrations 47.4, 80.7, 109.7, 193.5</li>
 +
</ul>
 +
 
 +
<p class="obj">Objective: Switch dCas9-tracrRNA into pSB1C3</p>
 +
<p>Treat pSB1C3 with Antarctic Phosphatase</p>
 +
<ul>
 +
<li>cleaned copy from 7/28 with 100 uL final volume</li>
 +
<li>2 hrs at 37C</li>
 +
</ul>
 +
 
 +
<p>PCR cleanup of AP-treated pSB1C3</p>
 +
<ul>
 +
<li>Qiagen kit</li>
 +
<li>concentration 104.5 ng/uL in 50 uL</li>
 +
</ul>
 +
 
 +
<p>Ligation of pSB1C3 and dCas9-tracrRNA</p>
 +
<ul>
 +
<li>2 experimental plates</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>3x molar dCas9-tracrRNA &ldquo;insert&rdquo;</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pSB1C3 100 ng = 1 uL</li>
 +
<li>dCas9-tracrRNA 600 ng = 7.5 uL</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>3x molar pSB1C3 &ldquo;backbone&rdquo;</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>pSB1C3 150 ng = 1.5 uL</li>
 +
<li>dCas9-tracrRNA 100 ng = 1.2 uL</li>
 +
</ul>
 +
 
 +
<ul>
 +
<li>Backbone-only and insert-only controls</li>
 +
<li>Transformed via heat shock and plated on LB+Cm</li>
 +
</ul>
</div>
</div>
</div>
</div>
 +
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Latest revision as of 15:11, 16 October 2014

July 1

Objective: make LB+Cm plates

  • Four sleeves of plates with 4x500mL medium
  • 34mg/mL chloramphenicol

Objective: insert crRNAs into pdCas9 and scaffold

Plate results

  • Experimental had lots of colonies, but backbone-only control did as well
  • reasoning: similarity of BsaI overhangs may lead to re-ligation
  • solution: treat with CIP before ligation

Agarose gel of BsaI cuts of pdCas9 and Repeat scaffold

  • plasmids used in 6/30/14 transformation
  • to make sure BsaI is cutting properly
  • Results: single band in both appears to be cut DNA

CIP treatment of pdCas9 and Repeat scaffold (both BsaI cut)

  • pdCas9: 10uL plasmid in 20 uL reaction with 1.5uL CIP
  • Repeat scaffold: 22uL plasmid in 30 uL reaction with 2 uL CIP
  • 2 hours at 37C (longer than standard protocol)

PCR cleanup of pdCas9 and Repeat scaffold CIP treatments

  • Final concentrations too low to be of use
  • Particularly negligible in pdCas9
  • Need to culture and cut more plasmid to try ligation again

Spread plates from pdCas9 and Repeat scaffold frozen stocks

  • Used tube “1” for repeat scaffold
  • Plated on fresh LB+Cm plates
  • Plates still slightly wet, difficult to spread evenly

Objective: make CCEC stock of DH5alpha-ZI strain

Diluted back 5mL culture in morning

  • 1mL into 5mL LB+Spec
  • note for future: Spec not necessary for most procedures, chromosomally integrated genes are more stable

Inoculated 1 mL culture into each of two 250mL flasks

  • Flasks contain SOC
  • 30C shaker for overnight growing

July 2

Objective: Make CCEC stock of DH5alpha-ZI strain

Made fresh CCMB-80 buffer stock

  • precipitate formed during pH adjusting and may have filtered out
  • could be detrimental to final product

Followed iGEM online protocol to make CCEC from Z1 strain

  • Overnight culture was much more concentrated than recommended
  • OD >1
  • Did not have enough SOC to dilute back fully, so diluted back only a bit
  • Had to vortex to resuspend cells at two stages of procedure
  • Final OD after procedure ~1.6 for 100mL of cells
  • Aliquoted 1mL into tubes, froze at -80C

Next steps:

  • Test competence with useful strains containing Lac, Tet promoters

Objective: insert crRNAs into pdCas9 and Repeat-scaffold

Culture colonies from plates of pdCas9 and pSB1C3-Repeat-scaffold

  • 3 colonies per plate in LB+Cm medium at 37C overnight
  • Plates were messy--hard to get individual colonies

July 3

Objective: insert crRNAs into pdCas9 and Repeat-scaffold

Miniprep cultures from 7/2

  • Three tubes of pdCas9 with concentrations 143.4, 145.7, 151.4 ng/uL
  • Three tubes of Repeat-scaffold with concentrations 166.6, 145.8, 159.6 ng/uL

Digest tubes 1 and 2 of pdCas9 and tubes 1 and 2 of Repeat-scaffold

  • to insert crRNAs
  • Digest with BsaI in cutsmart buffer
  • 50 uL DNA in 100 uL reactions with 7.5 uL BsaI
  • 3 hours 20 min at 37C

PCR cleanup of BsaI digest

  • final concentrations (in 30 uL each):
  • pdCas9: 65.4, 70.5 ng/uL
  • Repeat-scaffold: 122.3, 114.8 ng/uL

CIP treatment of pdCas9 and Repeat-scaffold BsaI digests

  • Two digest tubes pooled into one CIP tube for each backbone
  • 60 uL DNA in 100 uL reactions with 5 uL CIP in cutsmart buffer
  • 1.5 hours at 37C

PCR cleanup of CIP treatment

  • final concentrations (in 30 uL):
  • pdCas9: 32.5 ng/uL
  • Repeat scaffold 129.9 ng/uL

Annealed oligos of crRNA inserts for ligation

  • 3 crRNAs (GFP1, GFP2, GFP3) with 2 oligos each
  • 5uL each oligo heated to ~95C, slowly brought to rt in water bath

Ligation of crRNA inserts into pdCas9 and Repeat-scaffold

  • 6 working tubes + 5 controls
  • pdCas9 + each crRNA (GFP1, GFP2, or GFP3)
  • Repeat-scaffold + each crRNA (GFP1, GFP2, or GFP3)
  • pdCas9 Backbone only control
  • Repeat-scaffold Backbone only control
  • Insert only controls for each insert
  • 100ng = 3uL pdCas9, BsaI digested and CIP treated and cleaned
  • 100ng = 0.8uL Repeat-scaffold, BsaI digested and CIP treated and cleaned
  • 1uL annealed oligo insert
  • 10 uL reactions with 0.5 uL T4 Ligase
  • Transformed into DH5alpha and plated on LB+Cm

Objective: Test competency and effectiveness of DH5alpha-Z1 strain

CCEC Transformation efficiency test

  • Transformed 5 tubes of pSB1C3-K741002 into DH5alpha-Z1 CCEC
  • Total amount of DNA in 10 uL: 130ng, 13ng, 1.3ng, 0.13ng, and 0.013ng
  • Also for use in testing pLac regulation in Z1 strain

Transformed iGEM kit plate 3-6G into DH5alpha-Z1

  • contains pSB1C3-BBa_I13521: pTet-RBS-mRFP
  • in order to test regulation of pTet in Z1 strain

July 4

Objective: ligate crRNAs into pdCas9 and Repeat-scaffold

Plate results for ligation of crRNA GFP1, GFP2, and GFP3 into two backbones:

  • ~1000 colonies on all experimental plates
  • BO control for pdCas9 has ~500 colonies
  • BO control for Repeat scaffold has ~1000 colonies
  • IO controls all have 0 colonies

Cultured 4 copies of each crRNA-GFP1,2,3 in pdCas9 backbone

  • 12 tubes total in LB+Cm
  • None from Repeat-scaffold attempts

Objective: Test competency and effectiveness of DH5alpha-Z1 strain

CCEC Test plate results:

  • 130 ng: 100s of colonies
  • 13 ng:~100 colonies
  • 1.3 ng: ~20 colonies
  • 0.13 ng: 0 colonies
  • 0.013 ng: 0 colonies
  • Competency ~10 colonies/ng (low but working)

Results of transformation of pSB1C3-BBa_I13521 into DH5alpha-Z1

  • Plate had ~5 colonies

Cultured colonies for Tet and Lac testing in DH5alpha-Z1

  • One colony of pSB1C3-K741002
  • One colony of pSB1C3-BBa_I13521
  • In LB+Cm

July 5

Objective: Insert crRNAs into pdCas9 backbone

Saved stocks of 12 strains in 15% glycerol at -80C

  • 4 tubes each of potential GFP1,2,3 crRNA inserts in pdCas9

Miniprepped 12 tubes of pdCas9 with potential GFP-1,2,3 inserts

  • 4 tubes each
  • Eluted in 50 uL EB, all concentrations 100-200 ng/uL

Objective: Test effectiveness of DH5alpha-Z1 strain

Saved stock of pSB1C3-K741002 in DH5alpha (15% glycerol at -80C)

  • Culture of I13521 did not grow

Cultured another colony of pSB1C3-BBa_I13521

  • from 7/3 plate

July 6

Objective: Test effectiveness of DH5alpha-Z1 strain

Saved stock of pSB1C3-I13521 in DH5alpha (15% glycerol at -80C)

July 7

Objective: insert crRNAs GFP1,2,3 into pdCas9

Analytical restriction digest

  • 12 tubes with potential inserts (4 per crRNA)
  • pdCas9 as a control
  • BsaI and XbaI in cutsmart for 1.5 hrs

Agarose gel of restriction digest

  • Gel 1:
  • 1-4: pdCas9-crRNA-GFP1
  • 5-8: pdCas9-crRNA-GFP2
  • 9-12: pdCas9-crRNA-GFP3
  • Gel 2:
  • pdCas9 (control)
  • Expected single band ~9.3 kb if BsaI cut and ligation worked
  • Control and uncut failures expect two bands, 5.0 and 4.2 kb
  • Results: all 12 samples have single band
  • No uncut plasmids
  • Could still be recircularized with no insert

gel pictures:

Anneal oligos of 3 crRNA-GFP inserts

  • Following Dewran’s crRNA protocol
  • 2 uL each oligo diluted in PBS to 20 uL
  • Placed in 98C water bath, cooled gradually to rt

Phosphorylate oligo inserts of crRNAs

  • 5 uL annealed oligo mix in 20uL 1x ligase buffer with 1uL PNK
  • 30 mins at 37C, then deactivated for 20 mins at 65C
  • Diluted 1/50

Ligation

  • 75 ng backbone = 0.6 uL Repeat scaffold or 2.5 uL pdCas9
  • Both BsaI cut and CIP treated 7/3/14
  • 1.5 uL diluted, phosphorylated, annealed oligo insert
  • 6 experimental tubes + 2 BO controls and 3 IO controls
  • Transformed into iGEM CCEC (DH5alpha) and plated on LB+Cm

Cultured 2 colonies from pdCas9 plate

  • For Charlie to try his hands at ligation

Objective: Prepare various backbones for multiple-plasmid use

Transformed two distribution kit parts

  • Plate 4-6H: pSB4K5-J04450
  • backbone contains pSC101 origin with Kan resistance
  • Plate 4-18A: pSB3K3-I20260
  • backbone contains p15A origin with Kan resistance
  • On LB+G418 plates

Streaked plate from frozen stock

  • pSB6A1-J04450
  • backbone contains pMB1 origin with Amp resistance
  • on LB+Amp

Objective: Test pTet and pLac in DH5alphaZ1 strain

Streaked plates from frozen stock

  • pSB1C3-K741002 in DH5alpha-Z1
  • contains pLac-GFP
  • pSB1C3-I13521 in DH5alpha-ZI
  • contains pTet-RFP
  • On LB+Cm plates

July 8

Objective: insert crRNAs into pdCas9

Plate results from 7/7 transformation

  • pdCas9-crRNAs: ~50 colonies each
  • pdCas9 BO: 100s of colonies
  • Repeat-crRNAs: 100s of colonies
  • Repeat BO: ~1000 colonies
  • IO controls: 0 colonies, ~15 for crRNA-GFP3
  • Notes: unsure why BO controls have more colonies than experimental

Restriction digest of 12 samples from 7/5 prep + pdCas9 control

  • With SacI/NheI
  • Should show difference between insert and recircularized backbone

Made and ran 1.5% agarose gel in TBE

  • for better resolution of small band differences in pdCas9 with/without insert
  • 1: pdCas9 (control)
  • 2-5: pdCas9-crRNA-GFP1 (1-4)
  • 6-9: pdCas9-crRNA-GFP2 (1-4)
  • 10-13: pdCas9-crRNA-GFP3 (1-4)
  • 14: pdCas9 (control)
  • expected:
  • 3.1, 2.8, and 2.7 kb bands
  • 585 bp band with no insert, 620 bp band with insert (pdCas9 620bp)
  • Results: One sample, crRNA-GFP3-3, has larger band matching control
  • This colony may be correct, should be sequenced
  • Others have smaller band, are probably incorrect

Culture 40 colonies from existing plates to screen for crRNA inserts

  • 10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/3/14
  • 10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/7/14
  • in LB+Cm

Objective: Prepare various backbones for multiple-plasmid use

Prepared LB+Kan medium

  • 50 ug/mL final concentration Kanamycin

Cultured colonies from transformations and stock streaking

  • 3 colonies each from 3 plasmids:
  • pSB6A1-J04450 in LB+Amp
  • pSB3K3-I20260 in LB+Kan
  • pSB4K5-J04450 in LB+Kan

Objective: Test pLac and pTet in DH5alpha-Z1

Notes:

  • Need to obtain aTc for pTet testing
  • Tried to use Doxycycline, but had trouble with solubility in ethanol (?)
  • pLac is not the final promoter to be used, a different variant will be used
  • with no glucose dependency
  • Flow testing of pLac and pTet was suspended until these changes are made

July 9

Objective: Insert crRNAs into pdCas9

Miniprep 40 cultures of potential crRNA inserts in pdCas9

  • Labeled with three numbers
    • Date of transformation - crRNA-GFPx - sample number

700uL of each culture saved to freeze in glycerol if colonies appear successful

  • Stored at 4C for the day

Analytical restriction digest of potential crRNA inserts

  • All 40 tubes + 2 pdCas9 tubes as control
  • 1 uL DNA in 10 uL digest for 2 hrs at 37C
  • SacI-HF and NheI-HF in cutsmart

Made and ran 1.5% agarose/TBE gels

  • To differentiate between small band differences
  • Gel 1:
    • 1. 2-log ladder
    • 2. pdCas9
    • 3-12. pdCas9-crRNA-GFP1 from 7/3 transformation
    • 13-22. pdCas9 -crRNA-GFP2 from 7/3 transformation
    • 23. pdCas9
    • 24. 2-log ladder
  • Gel 2:
    • 1. 2-log ladder
    • 2. pdCas9
    • 3-12. pdCas9-crRNA-GFP1 from 7/7 transformation
    • 13-22. pdCas9 -crRNA-GFP2 from 7/7 transformation
    • 23. pdCas9
    • 24. 2-log ladder
  • Expected results:
    • pdCas9 and successful inserts:
      • 3.1, 2.8, and 2.7 kb bands, plus 620 bp insert band
    • unsuccessful recircularizations:
      • 3.1, 2.8, and 2.7 kb bands, plus 585 bp insert band
  • Results:
    • Gel 1 appears to only be recircularized
    • Gel 2 has some promising inserts:
      • GFP1 samples 1,2, and 3 from 7/7 transformation
      • GFP2 samples 7,8, and 9 from 7/7 transformation
  • Glycerol stocks frozen for these six samples
  • Next Steps:
    • Run digest with BsaI to confirm that plasmids are not uncut pdCas9
    • Sequence to confirm presence of insert (using oligos ordered 7/8/14)
Objective: Obtain new backbones and switch inserts into new backbones

Miniprep 3 new backbones

  • pSB6A1-J04450 (3 copies)
    • concentrations 437.6, 483.9, and 450.1 ng/uL
  • pSB3K3-I20260 (3 copies)
    • concentrations 114.6, 120.0, and 97.3 ng/uL
  • pSB4K5-J04450 (3 copies)
    • concentrations 129.3, 165.9, and 155.1 ng/uL

Prep-scale digest of all 9 backbone tubes

  • 50 uL DNA in 60 uL total reaction
  • EcoRI-HF/Spe-HF in cutsmart
  • 3 hours at 37C

Agarose gel extraction of 3 backbones

  • Gel 1, Left: pSB6A1
  • Gel 1, Right: pSB3K3
  • Gel 2, Left: pSB4K5
  • All 3 lanes had 2 bands as expected
    • upper band (backbone) in 3K3 had messy trail behind it (not extracted)
  • All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well
  • top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C

Streak plate from frozen stock

  • pSB1C3-K608012
  • In order to switch into pSB6A1
Objective: Remake new version of pDGC3

Transformation from iGEM distribution kit plates

  • Plate 2-6D: pSB1C3-BBa_R0011
    • Engineered Lac promoter (not affected by glucose)
  • Plate 3-15O: pSB1C3-BBa_I13500
    • Strong RBS-GFP
  • Plated on LB+Cm

July 10

Objective: Obtain backbones and switch parts into new backbones

Gel cleanup of 3 backbones with Zymoclean prep kit

  • Some tubes had to be divided into two samples for cleanup
  • Final elution was 20 uL each into 2-3 tubes per backbone
  • Nanodrops looked unclean: Most had high peak around 220-240 nm
    • pSB6A1: 300mg + 120 mg gel = 230.4 ng/uL
      • Peak ~220, then hump ~260
    • pSB6A1: 270 mg gel = 220 ng/uL
      • Peak, then hump as before
    • pSB3K3: 320 mg gel = 53.9 ng/uL
      • No clear sign of DNA in graph
    • pSB3K3: 230 mg gel = 20 ng/uL
      • No DNA
    • pSB3K3: 130 mg gel = 72 ng/uL
      • Possibly DNA, best option but may not be useful
    • pSB4K5: 290 mg gel = 43.4 ng/uL
      • No clear sign of DNA in graph
    • pSB4K5: 320 mg gel = 117.3 ng/uL
      • Possibly DNA, best option but may not be useful

Culture 3 colonies of pSB1C3-K608012

  • In order to switch into pSB6A1
Objective: Insert crRNAs into pdCas9

Analytical Digest of promising crRNA inserts

  • XbaI/BsaI digest
  • In order to confirm that plasmids are not uncut pdCas9
  • Samples 7-1-1,2,3 and 7-2-7,8,9 (6 tubes) plus pdCas9 as control

Agarose gel of digest results:

  • 1. pdCas9
  • 2-4. pdCas9-crRNA-GFP1, samples 1,2,3
  • 5-7. pdCas9-crRNA-GFP2, samples 7,8,9
  • Expected:
    • 2 bands at 4.2 and 5.1 kb in pdCas9
    • 1 band at 9.3 kb in successful plasmids with inserts
  • Results:
    • pdCas9 appears to be an incomplete digest, showing 2 small and 1 large band
    • Lack of any trace of smaller bands in 6 experimental samples indicates successful inserts.
Objective: Make pDGC3

Culture 3 colonies each from pSB1C3-R0011 and pSB1C3-I13500

Objective: Test effectiveness of DH5alpha-Z1 strain
Mitch and Sargis

Culture pSB1C3-I13521 (pTet-RFP)

  • For flow
  • In aTc and non-aTc

July 11

Objective: Switch K608012 into pSB6A1

Miniprep 3 copies of pSB1C3-K608012

  • concentrations 361, 324, 362 ng/uL

Prep-scale digest of pSB1C3-K608012

  • 2 miniprep tubes, 50 uL DNA in 100 uL each
  • With EcoRI and SpeI
  • 2+ hrs at 37C

Gel extraction and cleanup to isolate K608012

  • Cut lower band
  • Zymoclean prep kit
  • Each of 2 bands divided into two tubes during cleanup
    • 1A: 280 mg =
    • 1B: 240 mg =
    • 2A: 200 mg =
    • 2B: 200 mg = 62.5 ng/uL (20 uL)
  • Graphs on nanodrop did not look ideal: peak lower than usual
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Miniprep pSB1C3-R0011 and pSB1C3-I13500

  • 3 copies each
  • Concentrations
    • pSB1C3-R0011: 181, 229, 194 ng/uL
    • pSB1C3-I13500: 321.5, 228, 300.4 ng/uL

Prep scale digests of two tubes from each miniprep

  • pSB1C3-R0011 with SpeI/PstI
    • no extraction necessary
  • pSB1C3-I13521 with XbaI/PstI
    • to extract I13521 insert
  • 100 uL total reaction per tube
  • 2-3 hrs at 37C

Agarose gel of pSB1C3-I13500 XbaI/PstI digests

  • Intended for gel extraction
  • Expected 2 bands, but only one present
  • No extraction performed

Analytical agarose gel of pSB1C3-R0011 SpeI/PstI digests

  • Single band appears correct
  • see below for gel picture

PCR cleanup of pSB1C3-R0011 SpeI/PstI digests

  • concentrations 124.8, 157.3 ng/uL (30 uL each)
Objective: Switch dCas9-tracrRNA into pSB1C3

Miniprep pdCas9

  • concentration 167.4 ng/uL

Prep-scale digest of pSB1C3-R0011

  • 1 miniprep with XbaI/PstI
  • To extract pSB1C3 backbone

Agarose gel of pSB1C3-R0011 XbaI/PstI digest

  • Intended for gel extraction
  • Expected 2 bands, but only one present
  • No extraction performed

PCR of dCas9-tracrRNA and anti-tracrRNA

  • 4 tubes each, with pdCas9 as template in 0, 0.4, 0.7, and 1.0 uL quantities
  • oligos dCas9tracr-up and dCas9tracr-dn for dCas9-tracrRNA PCR
  • oligos dCas9tracr-up and tracrRNA-dn for anti-tracrRNA PCR
  • Q5 polymerase, with 64C annealing and 2 min extension

Agarose gel of PCR (and digest) results:

  • Lanes 1-4: dCas9-tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
  • Lanes 5-8: tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
  • Lanes 9-10: pSB1C3-R0011 SpeI/PstI digests
  • Results:
    • dCas9-tracr PCR appears correct, band ~4 kb in 3 lanes
    • tracrRNA PCR unclear: strong primer-sized band may be correct, faint bands ~4 kb indicate off-target extension.
      • tracrRNA PCR should be done with shorter extension time

PCR cleanup of dCas9-tracrRNA PCR

  • Concentration >200 ng/uL
Objective: Test BsaI and XbaI enzymes

Analytical digest of dCas9

  • One tube with BsaI/EcoRI
  • One tube with XbaI/EcoRI
    • XbaI from tube with exp. 5/14
  • One tube with XbaI/EcoRI
    • XbaI from tube with exp. 2/15

Agarose gel of digest:

  • 1. BsaI/EcoRI: expected 3 bands at 2.7, 2.9, and 3.5 kb
  • 2. XbaI (5/14) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
  • 3. XbaI (2/15) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
  • Results: Lanes 1 and 3 look as expected. Lane two has one extra band at 3.5 kb corresponding to partial digestion with XbaI. 5/14 XbaI tube is ineffective and has been thrown out

July 14

Objective: insert crRNAs into pdCas9

Sequencing Order #1608

    • BigDye reaction with BD buffer 1.1
    • pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9up1
    • pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9up1
    • pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9up1
    • pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9up1
    • pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9up1
    • pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9up1
    • pdCas9-crRNA_GFP3 sample 3 w/ pdCas9up1
    • pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9dn1
    • pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9dn1
    • pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9dn1
    • pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9dn1
    • pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9dn1
    • pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9dn1
    • pdCas9-crRNA_GFP3 sample 3 w/ pdCas9dn1

Results:

    • pdCas9-crRNA_GFP1 use sample 7-1
    • pdCas9-crRNA_GFP2 use sample 7-9
    • pdCas9-crRNA_GFP3 need to find more colonies
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Prep scale digest of pSB1C3-I13500 with XbaI/PstI

  • Tube 3 from 7/11/14 miniprep
  • To extract both I13500 and pSB1C3 backbone
  • 100 uL reaction with 50 uL DNA in cutsmart

Agarose gel extraction and cleanup of pSB1C3 and I13500

  • Top band (~2kb): pSB1C3
  • Bottom band (~1kb): I13500
  • Zymoclean prep kit
  • Concentrations:
    • pSB1C3: 319 ng/uL in 20 uL (dirty)
    • I13500: 91.2 ng/uL in 20 uL (dirty)

Ligation of pSB1C3-R0011 and I13500

  • 100ng = 0.7 uL pSB1C3-R0011 (#2) cut with SpeI/PstI on 7/11
  • 150ng = 1.8 uL I13500 cut with XbaI/PstI on 7/14
  • Experimental, BO control, and IO control
  • rt for 1 hr
  • Heat shock transformation into DH5alpha and plated on LB+Cm
Objective: Switch K608012 into pSB6A1

Ligation of pSB6A1 and K608012

  • 100 ng = 0.5 uL pSB6A1 (“Yellow” sample) cut with EcoRI/SpeI on 7/9
  • 150 ng = 2.5 uL K608012 (#2B) cut with EcoRI/SpeI on 7/11
  • Experimental, BO control, and IO control
  • rt for 1 hr
  • Heat shock transformation into DH5alpha and plated on LB+Amp
Objective: Switch dCas9-tracrRNA into pSB1C3

Prep scale digests

  • dCas9-tracr PCR with XbaI/PstI (no extraction)
  • pSB1C3-K608012 (tube 3) with XbaI/PstI (to obtain pSB1C3)
  • 100 uL reaction each with 50 uL DNA in cutsmart

PCR cleanup of dCas9-tracr digest

  • Qiagen miniprep kit
  • Concentration 38.1 ng/uL in 30 uL

Agarose gel extraction and cleanup of pSB1C3

  • gel order: left K608012, right I13500
  • extracted pSB1C3, top band of K608012 (~2 kb)
  • Zymoclean kit
  • Concentration 127.9 ng/uL in 20 uL (dirty)

Ligation of pSB1C3 and dCas9-tracrRNA PCR

  • 100 ng = 3 uL dCas9-tracrRNA PCR cut with XbaI/PstI on 7/14
  • 150 ng = 0.6 uL pSB1C3 (from I13500) cut with XbaI/PstI on 7/14
  • Experimental, BO control, and IO control
  • rt for 1 hr
  • Heat shock transformation into DH5alpha and plated on LB+Cm
Objective: Create pTet-anti-tracrRNA

Prep-scale digest of pSB1C3-R0040

  • With SpeI/PstI (no extraction necessary)
  • 100 uL reaction with 50 uL DNA in cutsmart
  • 4 hrs at 37C

PCR anti-tracrRNA from pdCas9

  • Oligos dCas9tracr-up and tracrRNA-dn
  • 0, 0.4, 0.7, and 1.0 uL pdCas9 template in 50 uL reactions
  • Q5 polymerase
  • annealed at 64C, 20 sec extension

Agarose gel of anti-tracrRNA PCR

  • Results: only 1.0 uL template lane appeared to amplify
    • Need to make more using this template concentration

PCR anti-tracrRNA from pdCas9

  • Oligos dCas9tracr-up and tracrRNA-dn
  • 1.0 uL pdCas9 template in each 50 uL reaction
  • 8 tubes with Q5 polymerase
  • annealed at 64C, 20 sec extension

Agarose gel of anti-tracrRNA PCR and pSB1C3-R0040 digest

  • Lanes 1-8: anti-tracrRNA PCR
  • Lane 9: R0040 digest
  • Results:
    • PCR appears to work in all lanes (band size ~0.1 kb)
    • R0040 digest single band appears correct

PCR cleanup of anti-tracrRNA PCR and pSB1C3-R0040

  • Concentrations:
    • anti-tracrRNA: 83.3 ng/uL in 30 uL
    • pSB1C3-R0040: 25 ng/uL in 30 uL

July 15

Objective: Create pTet-anti-tracrRNA

Digest of anti-tracrRNA PCR

  • with XbaI, PstI-HF, and DpnI
  • 3 hrs at 37C
  • 60 uL DNA in 100 uL total reaction

PCR Cleanup of anti-tracrRNA PCR digest and pSB1C3-R0040 digest

  • Concentrations
    • anti-tracrRNA: 100 ng/uL
    • pSB1C3-R0040: 35 ng/uL

Ligation of anti-tracrRNA and pSB1C3-R0040

  • 100 ng = 3 uL pSB1C3-R0040 cut with SpeI/PstI
  • 15 ng = 0.15 uL anti-tracrRNA PCR cut with XbaI/PstI/DpnI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Cm
Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • Experimental: 8 colonies
  • BO Control: 4 colonies
  • IO Control: 7 colonies

Culture colonies

  • 4 copies of potential pSB1C3-dCas9-tracrRNA construct

Digest of dCas9-tracrRNA PCR and heat inactivation

  • Digest with DpnI
  • 22 uL DNA in 30 uL reaction
  • 1 hr at 37C, then 30 mins at 80C to heat-inactivate
  • assumed concentration 20 ng/uL

PCR Cleanup of pSB1C3 digest

  • 2 tubes of gel-extracted and cleaned pSB1C3 cut with XbaI and PstI
  • 11 ug original yields 309.6 ng/uL in 20 uL “superclean”

Ligation of dCas9-tracrRNA PCR and pSB1C3

  • 150 ng = 0.5 uL pSB1C3 cut with XbaI and PstI
  • 100 ng = 5 uL dCas9-tracrRNA PCR cut with Xbal/PstI/DpnI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Cm
Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block

Plate results:

  • Lawn of colonies on Experimental and BO control
  • No colonies on IO control

Digest of pSB1C3-R0011

  • With SpeI, PstI, and CIP
  • 50 uL DNA in 100 uL reaction
  • 2.5 hrs at 37C

PCR Cleanup of pSB1C3-R0011 digest and I13500 digest

  • pSB1C3-R0011 concentration 175 ng/uL
  • I13500 (from previous digestion and extraction)
    • 2.3 ug original yields 124 ng/uL in 20 uL “superclean”

Ligation of I13500 and pSB1C3-R0011

  • 100 ng = 0.6 uL pSB1C3-R0011 cut with SpeI/PstI/CIP
  • 150 ng = 1.2 uL I13500 cut with XbaI/PstI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Cm
Objective: Switch K608012 into pSB6A1

Plate results:

  • 2 colonies on experimental plate
  • 1 colony on BO, no colonies on IO

Cultured colonies

  • 2 colonies of potential pSB6A1-K608012 construct

PCR Cleanup of pSB6A1 digest and K608012 digest

  • pSB6A1, 2 tubes gel extracted and cleaned
    • 10 ug original yields 203 ng/uL in 30 uL
  • K608012, 3 tubes gel extracted and cleaned
    • 4 ug original yields 73.0 ng/uL in 30 uL

Ligation of K608012 and pSB6A1

  • 100 ng = 0.5 uL pSB6A1 cut with EcoRI and SpeI
  • 150 ng = 2 uL K608012 cut with EcoRI and SpeI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Amp

July 16

Objective: Create pTet-anti-tracrRNA

Plate results:

  • Experimental: over 1000 colonies
  • BO control had ~500 colonies, IO control had ~50 colonies

Cultured colonies

  • 4 copies of potential pSB1C3-R0040-anti-tracrRNA
Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • Experimental: 12-15 colonies
  • BO control had 3 colonies, IO control had ~30 colonies

Minipreps

  • 4 copies of potential pSB1C3-dCas9-tracrRNA construct
  • From 7/14 ligation
  • concentrations: 537.2, 45.4, 79.1, and 71.5 ng/uL
    • Only one has normal concentration, others very low

Analytical digest of pSB1C3-dCas9-tracrRNA constructs:

  • 4 copies with NheI/XbaI

Agarose gel of digests:

  • Lanes 1-2: pSB6A1-K608012 with MfeI/XbaI (expected 4.1, 0.6 kb bands)
  • Lanes 3-6: pSB1C3-dCas9-tracrRNA with NheI/XbaI (expected 5.0, 1.5 kb bands)
  • Results: gel was fuzzy and unclear
    • lanes 2,3,5,6 clearly incorrect: 1 band ~2.0 kb
    • lane 1 had one fuzzy band around 4-5 kb (could be incomplete digest)
    • lane 4 had band around 5 kb with smear above (could be incomplete digest)

New analytical digest of pSB1C3-dCas9-tracrRNA construct

  • Only copy #2 (showed potential in previous digest
  • With KpnI/SpeI for 2 hours at 37C
  • Also put previous digest back in bath for 2 more hours

Agarose gel of digests

  • DETAILS
Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block

Plate results:

  • ~500 colonies on experimental plate
  • BO control had over 1000 colonies
  • IO control had no colonies

Cultured colonies

  • 4 copies of potential pSB1C3-R0011-I13500 construct
Objective: Switch K608012 into pSB6A1

Plate results:

  • 0 colonies on experimental plate
  • 4 colonies on BO control, 1 colony on IO control
Objective: Insert crRNAs into pdCas9

Colony PCR of pdCas9-GFP3 plate

  • Plate from 7/7/14
  • 15 colonies plus pdCas9 miniprep as control
  • double dipped in 50 uL Taq poly mix and 50 uL SOC
  • Oligos pdCas9-up1 and pdCas9-dn1
  • TALCOLONYPCR protocol with 64C anneal

Agarose gel of colony PCR

  • Expected to see higher band matching control in successful colonies
  • lanes 1 and 17 are pdCas9 control
  • assay looks helpful, successes in samples 3,5,6,8,9,12,13,15

Cultured colonies

  • Copies 3,5,6, and 8 from colony PCR in LB+Cm

July 17

Objective: Create pTet-anti-tracrRNA and pSB1C3-R0011-I13500

Minipreps

  • 4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation
  • 4 copies of pSB1C3-R0011-I13500 from 7/15 ligation

Analytical digest of minipreps

  • pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI
  • pSB1C3-R0011-I13500 with MfeI/NdeI/SacI

Agarose gel of digest results:

  • Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, <0.1 kb bands)
  • Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)
  • Results:
  • R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly
  • R0011-I13500 does not look correct (only one band at 2.0 kb)
Objective: Switch dCas9-tracrRNA into pSB1C3

Colony PCR on pSB1C3-dCas9-tracrRNA plate

  • Ligation plate from 7/15/14
  • 15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC
  • oligos SB1C3-up1 and SB1C3-dn1
  • annealed at 65C, extension 2 mins

Agarose gel of colony PCR

  • results: primer-bands only (no successes)
Objective: Switch K608012 into pSB6A1

Culture colonies

  • 2 copies of pSB6A1-J04450 (from 6-9-14 plate)
  • 2 copies of pSB1C3-K608012

July 18

Objective: Switch dCas9-tracrRNA into pSB1C3

PCR of dCas9-tracrRNA from pdCas9

  • 8 tubes, each with 1 uL pdCas9 template at 1 ng/uL
  • oligos dCas9tracr-up and dCas9-tracr-dn
  • 2 min extension, 64C anneal

Agarose gel of PCR product

  • All 8 tubes look successful (band ~4 kb)

PCR cleanup of dCas9-tracrRNA

  • Qiagen kit
  • Into 2 tubes, 30 uL each

Prep-scale digest of PCR product

  • with PstI/XbaI/DpnI
  • in 100 uL each for 3 hrs at 37C

Treat pSB1C3 digest with CIP

  • 1 hr at 37C

Objective: Switch K608012 into pSB6A1

Miniprep pSB1C3-K608012 and pSB6A1-J04450

  • 1 copy each (the second copy of each did not grow in culture)
  • concentrations:
  • pSB1C3-K608012: 344 ng/uL
  • pSB6A1-J04450: 421.5 ng/uL

Prep-scale digest of K608012 and pSB6A1

  • 50 uL DNA in 100 uL reaction
  • Both with EcoRI and PstI
  • 3 hrs at 37C

CIP treatment of pSB6A1 and heat-inactivation

  • 2.5 uL CIP added to digest tube
  • Both digests returned to 37C for 1 hr
  • Then 30 mins at 80C to heat-inactivate

Gel extraction of pSB6A1 and K608012

  • Gel had no EtBr added, and was discarded (product was lost)

July 20

Objective: culture colonies for various purposes

Cultured colonies

  • 4 copies pSB6A1-J04450
  • 2 from older plate, 2 from newer
  • 4 copies pSB1C3-K608012
  • 2 from older plate, 2 from newer
  • 4 copies pSB1C3-B0030
  • To obtain pSB1C3 without gel extraction
  • 4 copies pSB1C3-R0011
  • 4 copies pSB1C3-I13500

July 21

Objective: Sequence to confirm constructs

Sequencing reaction and Order #1634

  • 1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1
  • 5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1
  • 9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1
  • 11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1
  • Big Dye, buffer version 1.1

Order 1634 Results:

  • pdCas9-GFP3 copies 3, 6, and 8 are correct
  • copy 5 has a single mutation
  • anti-tracrRNA: both copies are correct
  • copy 1 was a cleaner, more reliable

Objective: Create pSB1C3-R0011-I13500

Minipreps

  • Qiagen kit
  • 4 tubes of pSB1C3-R0011
  • concentrations 255.7, 169.2, 204.8, 151.2 ng/uL
  • 4 tubes of pSB1C3-I13500
  • concentrations 148.9, 214.2, 260.7, 432.5

Prep scale digests of pSB1C3-R0011 and I13500

  • pSB1C3-R0011 with SpeI/PstI, samples 1,3
  • pSB1C3-I13500 with XbaI/PstI, samples 1,3
  • 50 uL DNA in 100 uL each reaction, 2 tubes per construct
  • 3 hrs at 37C
  • Added CIP (2.5 uL) to pSB1C3-R0011 reaction
  • 1 more hr for both at 37C
  • Heat inactivation of both reactions: 30 mins at 80C

Gel extraction of I13500

  • extraction gel 1, lanes 1 and 2
  • Zymoclean prep kit
  • sample 1 (lane 1): 300 mg gel yields
  • sample 3 (lane 2): 550 mg gel yields

PCR cleanup of pSB1C3-R0011

  • Qiagen kit
  • Two tubes of digest combined into one tube product
  • Concentration

Ligation of pSB1C3-R0011 and I13500

  • Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL
  • New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL
  • Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL
  • New insert (I13500 extracted 7/21): 300 ng = 3 uL
  • Ligations numbered 1-8:
  • 1. OB+OI
  • 2. OB+NI
  • 3. NB+OI
  • 4. NB+NI
  • 5. OB control
  • 6. OI control
  • 7. NB control
  • 8. NI control
  • 1 hr at rt
  • Transformed via heat shock into DH5alphas, plated on LB+Cm

Objective: Switch dCas9-tracrRNA into pSB1C3

Minipreps

  • Qiagen kit
  • 4 tubes of pSB1C3-B0030
  • to obtain pSB1C3 without gel extraction
  • concentrations 116.8, 99.9, 115.3, 145.1 ng/uL

PCR cleanup of dCas9-tracrRNA PCR

  • Product digested 7/18 with XbaI/PstI/DpnI
  • Concentration 80.5 ng/uL in 30 uL
  • Heat inactivation of cleaned product, 30 mins at 80C

Prep scale digest of pSB1C3-B0030

  • With XbaI/PstI, samples 1,3
  • 50 uL DNA in 100 uL each reaction, 2 tubes
  • 3 hrs at 37C
  • Added CIP (2.5 uL)
  • 1 more hr for both at 37C
  • Heat inactivation: 30 mins at 80C

PCR cleanup of pSB1C3

  • B0030 should not be retained
  • Qiagen kit
  • Two tubes of digest combined into one tube product
  • Concentration

Ligation of pSB1C3 and dCas9-tracrRNA

  • Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL
  • Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL
  • New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL
  • Ligations numbered 9-13:
  • 9. B+OI
  • 10. B+NI
  • 11. B control
  • 12. OI control
  • 13. NI control
  • 1 hr at rt
  • Transformed via heat shock into DH5alphas, plated on LB+Cm

Objective: Switch K608012 into pSB6A1

Minipreps

  • Qiagen kit
  • 4 tubes of pSB6A1-J04450
  • concentrations 189.3, 432.5, 688.7, 487.3 ng/uL
  • 4 tubes of pSB1C3-K608012
  • concentrations 68.8, 91.6, 346.1, ng/uL

Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012

  • pSB6A1-J04450 with XbaI/SpeI, samples 1,3
  • pSB1C3-K608012 with EcoRI/PstI, samples 1,3
  • 50 uL DNA in 100 uL each reaction, 2 tubes per construct
  • 3 hrs at 37C
  • Added CIP (2.5 uL) to pSB6A1-J04450 reaction
  • 1 more hr for both at 37C
  • Heat inactivation of both reactions: 30 mins at 80C

Gel extraction of pSB6A1 and K608012

  • K608012: gel 1, lane 3 and gel 2, lane 1
  • pSB6A1: gel 2, lanes 2 and 3
  • Zymoclean prep kit
  • K608012 sample 1 (lane 1-3): 300 mg gel yields
  • K608012 sample 3 (lane 2-1): 550 mg gel yields
  • pSB6A1 sample 1 (lane 2-2):
  • pSB6A1 sample 3 (lane 2-3):

Ligation of pSB6A1 and K608012

  • Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL
  • Old insert (K608012 extracted 7/15): 300 ng = 4 uL
  • Ligations numbered 14-16:
  • 14. B+I
  • 15. B control
  • 16. I control
  • 1 hr at rt
  • Transformed via heat shock into DH5alphas, plated on LB+Amp

Gibson of pSB6A1 and K608012

  • New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
  • New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
  • Numbered 17-19:
  • 17. B+I
  • 18. B control
  • 19. I control
  • 15 uL Mert’s DIY Gibson mix
  • 1 hr at 60C
  • Transformed via heat shock into DH5alphas, plated on LB+Amp

SLIC of pSB6A1 and K608012

  • New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
  • New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
  • Numbered 20-22:
  • 20. B+I
  • 21. B control
  • 22. I control
  • 2.5 mins at rt, then 10 mins at 4C
  • Transformed via heat shock into DH5alphas, plated on LB+Amp

July 22

Objective: Create pSB1C3-R0011-I13500

Plate results:

  • All four experimental plates had lots of colonies
  • both Backbone-only controls had similar colony numbers to experimentals
  • Insert-only controls had no colonies

Colony PCR

  • 4 copies each of 4 experimental plates = 16 colonies
  • Oligos SB1C3-up and SB1C3-dn with Taq polymerase
  • 66C anneal temp, 2 min extension time

Agarose gel of colony PCR results

  • 1-4: OB+OI pSB1C3-R0011-I13500
  • 5-8: OB+NI pSB1C3-R0011-I13500
  • 9-12: NB+OI pSB1C3-R0011-I13500
  • 13-16: NB+NI pSB1C3-R0011-I13500
  • 17-20: B+OI pSB1C3-dCas9-tracrRNA
  • 21-24: B+NI pSB1C3-dCas9-tracrRNA
  • Results: no successful bands on any colonies
  • Just primer-dimers or small amplified bands?

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • Both experimental plates had lots of colonies
  • Backbone-only controls had similar colony numbers to experimentals
  • Insert-only controls had no colonies

Colony PCR

  • 4 copies each of 2 experimental plates = 8 colonies
  • Oligos SB1C3-up and SB1C3-dn with Taq polymerase
  • 66C anneal temp, 2 min extension time

Agarose gel of colony PCR results

  • see above for gel order
  • Results: no successful bands on any colonies
  • Just primer-dimers or small amplified bands?

Objective: Switch K608012 into pSB6A1

Plate results:

  • No colonies on Gibson or SLIC plates
  • 3 colonies on Ligation plate
  • A few colonies on controls, but not many

Cultured colonies

  • 3 colonies of potential pSB6A1-K608012 from Ligation plate
  • in LB+Amp

Ligation of K608012 and pSB6A1

  • Same constructs as 7/21 ligation
  • This time let recover in SOC for 2 hrs instead of 1 hr
  • Plated on LB+Amp

Objective: Try putting K608012 into pSB1A2 instead

Transformation from kit plate

  • Plate 4-1N
  • pSB1A2-B0034
  • Plated on LB+Amp

July 23

Objective: Switch K608012 into pSB6A1

Miniprep 3 copies of potential pSB6A1-K608012

  • Qiagen kit
  • Saved stocks in 15% glycerol

Analytical digest of minipreps

  • With NdeI in Cutsmart, 10 uL final volume

Agarose gel of analytical digest

  • Results:
  • Copies 1 and 2 are strange and incorrect
  • Copy 3 looks correct
  • Need to check with more digests

Analytical digest to check promising sample

  • Copy 3 in 3 different reactions
  • EcoRI/SpeI
  • EcoRI/MfeI
  • NcoI/PvuI
  • Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI
  • Control: pSB1C3-K608012 with MfeI/NcoI

Agarose gel of analytical digest

  • 1. Ligation 3 with EcoRI/SpeI
  • 2. Ligation 3 with EcoRI/MfeI
  • 3. Ligation 3 with NcoI/PvuI
  • 4. pSB6A1-J04450 with EcoRI/SpeI/PvuI
  • 5. pSB1C3-K608012 with MfeI/NcoI
  • Results:
  • Sample looks good
  • Incorrect samples 1 and 2 glycerol stocks thrown out

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Treat pSB4K5 with CIP

  • 1.25 uL CIP in 25 uL reaction
  • pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9

Prep-scale digest to obtain R0040-anti-tracrRNA

  • pSB1C3-R0040-anti-tracrRNA
  • with EcoRI/SpeI

Gel extraction and cleanup of R0040-anti-tracrRNA

  • Extracted smaller band
  • Attempted to clean up using Qiagen prep kit
  • dissolved with Zymoclean ADB
  • Then followed Qiagen PCR cleanup protocol
  • 510 mg gel yielded little or no DNA

Objective: Create pSB1C3-R0011-I13500

Colony PCR

  • 8 colonies from 7/21 ligation plates
  • Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011

Agarose gel of colony PCR results

  • No successes
  • Controls did not work properly either
  • colony PCR does not seem to be effective--need to just pick colonies

Culture colonies

  • 8 colonies of potential pSB1C3-R0011-I13500
  • From 7/21 ligation plates (colonies from today’s colony PCR)

Objective: Measurement interlab study

Transformations from kit plates

  • Plate 1-20K
  • pSB1C3-K823005
  • Plate 1-22I
  • pSB1C3-K823012
  • Plate 2-24B
  • pSB1C3-E0240

July 24

Objective: Create pSB1C3-R0011-I13500

Miniprep 8 colonies of potential pSB1C3-R0011-I13500

  • Qiagen kit

Analytical digest of minipreps

  • 8 copies with NcoI
  • 8 copies with EcoRI/SpeI

Agarose gel of digest results

  • 1-8: copies 1-8 with NcoI
  • 9-16: copies 1-8 with EcoRI/SpeI
  • Results: no successes, insert appears to be missing
  • Unsure whether incorrect colonies are pSB1C3 or pSB1C3-R0011

Agarose gel of digest results

  • Thicker gel, longer run time, more DNA
  • from same digest tubes
  • Results:
  • no small band on EcoRI/SpeI cut and no difference in larger band for the two cuts, indicating that R0011 is not present

Ligation of pSB1C3-R0011 and I13500

  • Used lab stock of T4 Ligase and buffer rather than iGEM stock
  • 100 ng pSB1C3-R0011 = 2.5 uL
  • 300 ng I13500 = 3 uL
  • 4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
  • BO and IO controls for each time
  • Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)

Objective: Switch dCas9-tracrRNA into pSB1C3

Ligation of pSB1C3 and dCas9-tracrRNA

  • Used lab stock of T4 Ligase and buffer rather than iGEM stock
  • 300 ng pSB1C3 = 3 uL
  • 100 ng dCas9-tracrRNA PCR = 1 uL
  • 4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
  • BO and IO controls for each time
  • Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)

Objective: Test GFP repression by crRNAs

Transform pSB6A1-K608012 into DH5alpha-ZI

  • Plated on LB+Amp

Objective: Obtain constructs for various uses

Streaked plate from frozen stock

  • One LB+Cm plate in thirds:
  • pSB1C3-R0040-antitracrRNA
  • pSB1C3-Repeat-seq scaffold
  • pSB1C3-Csy4-gRNA scaffold

Objective: Measurement interlab study

Cultured colonies (2 each)

  • pSB1C3-E0240
  • pSB1C3-K823005
  • pSB1C3-K823012
  • pSB3K3-I20260

July 25

Objective: Create pSB1C3-R0011-I13500

Plate results:

  • no apparent difference between 15, 30, 45, or 60 minute ligations
  • many colonies on all Experimental and BO control plates
  • no colonies on IO control

Colony PCR:

  • 16 colonies from pSB1C3-R0011-I13500 plates
  • 4 from each ligation time
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • 66C anneal temp, 45 sec extension

Agarose gel of colony PCR

  • No successes
  • It seems as though colony PCRs are not working--try control with a gradient

Gradient Colony PCR

  • to optimize with SB1C3-up and SB1C3-dn primers
  • template colonies of pSB1C3-K608011 from plate
  • anneal temps 62-72 C, extension time 45 sec

Agarose gel of gradient colony PCR

  • left to right: 62C to 72C anneal temps
  • Results: 62C was the best anneal temp--worked well
  • gradual decrease as temp increased, no amplification above 65C

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • no apparent difference between 15, 30, 45, or 60 minute ligations
  • many colonies on all Experimental and BO control plates
  • no colonies on IO control

Colony PCR:

  • 16 colonies from pSB1C3-dCas9-tracrRNA plates
  • 4 from each ligation time
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • 66C anneal temp, 45 sec extension

Agarose gel of colony PCR

  • No successes
  • It seems as though colony PCRs are not working--try control with a gradient
  • see gradient results above

Objective: Various ligations

Prep-scale digests

  • pSB1C3-Csy4-scaffold (2 copies) with XbaI/PstI
  • to ligate into R0040
  • pSB1C3-Repeat-seq scaffold (2 copies) with XbaI/PstI
  • to ligate into R0040
  • pSB1C3-R0040-anti-tracrRNA (2 copies) with XbaI/PstI
  • 100 uL reactions each tube

PCR cleanups on all digests

  • Qiagen kit
  • Concentrations (in 50 uL each):
  • pSB1C3-Csy4-scaffold: 55.9, 105.5 ng/uL
  • pSB1C3-Repeat-seq scaffold: 15.2, 55.2 ng/uL
  • pSB1C3-R0040-anti-tracrRNA: 90.6 ng/uL
  • Need to extract from gels before using, only cleaned up for safer storage

Objective: Measurement interlab study

Minipreps

  • Qiagen kit
  • pSB1C3-K823005 (2 copies)
  • concentrations 256.3, 314.8 ng/uL
  • pSB1C3-K823012 (2 copies)
  • concentrations 204.4, 192.5 ng/uL
  • pSB1C3-E0240 (2 copies)
  • concentrations 266.3, 241.5 ng/uL

Prep-scale digests

  • pSB1C3-K823005 (2 copies) with SpeI/PstI
  • pSB1C3-K823012 (2 copies) with SpeI/PstI
  • pSB1C3-E0240 (2 copies) with XbaI/PstI
  • 100 uL each tube

PCR cleanups on all digests

  • Qiagen kit
  • Concentrations (in 50 uL each):
  • pSB1C3-K823005: 156.3, 181.7 ng/uL
  • pSB1C3-K823012: 110.0, 98.0 ng/uL
  • pSB1C3-E0240: 110.0, 114.6 ng/uL

Objective: Test crRNA repression of GFP reporter

Cultured colony

  • To make CCEC of reporter strain
  • One colony of pSB6A1-K608012 in DH5alpha-ZI
  • Grew for 8 hrs during the day in 2 mL LB+Amp at 37C
  • Inoculated into 100 mL flask of LB+Amp for overnight shaker at rt

Flow cytometry of reporter strain

  • quick check to make sure reporter still works with new backbone
  • Used parameters from previous GFP flow data
  • Levels slightly under what was seen before, but still strong

July 26

Objective: Test crRNA repression of GFP reporter

Prepared Chemically competent E. coli

  • Using DH5alpha-ZI + pSB6A1-K608012
  • iGEM CCEC protocol with smaller volumes (made ~20 1-mL aliquots)

Double-Transformation of pdCas9 variants into reporter cells

Charlie Cooper

  • pdCas9 (with BsaI insert as crRNA sequence)
  • pdCas9-crRNA_GFP1
  • pdCas9-crRNA_GFP2
  • pdCas9-crRNA_GFP3

Objective: Create pSB1C3-R0011-I13500

Colony PCRs

  • 16 colonies from 7/24 ligation plates
  • oligos SB1C3-up and SB1C3-dn
  • 62C anneal (as determined by gradient), 45 sec extension

Agarose gel of Colony PCRs

  • Results: colonies 5 and 14 amplified at the correct size

Culture colonies

  • Colonies 5 and 14 from colony PCR of potential pSB1C3-R0011-I13500

Objective: Create pSB1C3-dCas9-tracrRNA

Colony PCRs

  • 16 colonies from 7/24 ligation plates
  • oligos SB1C3-up and SB1C3-dn
  • 62C anneal (as determined by gradient), 45 sec extension
  • PCRs left in machine without starting reaction for ~1 hr, could have kill reaction

Agarose gel of Colony PCRs

  • No successes

July 27

Objective: Test crRNA repression of GFP reporter

Cultured colonies for flow

  • 3 copies of each
  • DH5alpha-Z1 (background)
  • DH5-alpha-Z1 + pSB6A1-K608012 (reporter positive control)
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9 (non-target control)
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP1
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP2
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP3

Objective: Create pSB1C3-R0011-I13500

Minipreps

  • Colonies 5 and 14 from 7/26 colony PCRs
  • Qiagen kit

July 28

Objective: Create pSB1C3-R0011-I13500

Analytical digest of minipreps

  • Colonies 5 and 14 of potential pSB1C3-R0011-I13500
  • with MfeI
  • with NcoI
  • with EcoRI/PstI

Agarose gel of analytical digest results

  • Gel Order:
  • #14 w/ MfeI
  • #14 w/ EcoRI/PstI
  • #14 w/ NcoI
  • #5 w/ MfeI
  • #5 w/ EcoRI/PstI
  • #5 w/ NcoI
  • Results: All looks correct, except that MfeI cut once instead of twice
  • could be pSB1C3-I13500 without R0011

Objective: Insert mCherry G-block into pSB1C3-R0011-I13500

PCR of mCherry G-block

  • Q5 polymerase with Oligos SB1C3-dn and pDGC3-up
  • 4 tubes with 0, 0.4, 0.7, 1.0 uL template at 1 ng/uL
  • 45 sec extension, 66C anneal temp

Agarose gel of PCR

  • 3 tubes look successful

PCR cleanup of mCherry G-block PCR

  • Qiagen kit
  • 40 uL at 27.6 ng/uL

Objective: Various ligations

Gel extraction and cleanup

  • Each extraction included 2 cleaned digests combined into one lane (~100 uL)
  • Cut and cleaned with Zymoclean prep kit
  • pSB1C3-Csy4-scaffold cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 47.5 ng/uL in 30 uL
  • Csy4-scaffold insert 15.4 ng/uL in 30 uL
  • pSB1C3-Repeat-seq scaffold cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 74.8 ng/uL in 30 uL
  • Repeat-seq scaffold insert 13.1 ng/uL in 30 uL
  • pSB1C3-R0040-anti-tracrRNA cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 164.0 ng/uL in 30 uL
  • R0040-anti-tracrRNA 176.0 ng/uL in 30 uL

PCR Cleanup on gel extraction cleanups

  • to “superclean”
  • pSB1C3 4 tubes combined into 1: 469.6 ng/uL in 20 uL
  • Repeat-seq scaffold: 23.9 ng/uL
  • rough graph, probably not useful
  • Csy4 scaffold: 16 ng/uL
  • rough graph, probably not useful
  • R0040-anti-tracrRNA: 7.4 ng/uL
  • rough graph, probably not useful

Objective: Measurement interlab study

CIP treatment of backbones

  • Using Charlie’s CIP stock
  • pSB1C3-K823005 cut with SpeI/PstI on 7/25 (2 tubes)
  • pSB1C3-K823012 cut with SpeI/PstI on 7/25 (2 tubes)

Gel extraction and cleanup

  • 2 cleaned digests combined into one lane (~100 uL)
  • Cut and cleaned with Zymoclean prep kit
  • pSB1C3-E0240 cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 116.4 ng/uL in 30 uL
  • E0240 86.5 ng/uL in 30 uL

PCR Cleanup of CIP treatment and gel extraction cleanup

  • Qiagen kit
  • pSB1C3-K823005 SpeI/PstI CIP treated: 119.8, 119.7 ng/uL
  • pSB1C3-K823012 SpeI/PstI CIP treated: 60.9, 62.3 ng/uL
  • E0240 XbaI/PstI extracted: 56.5 ng/uL in 20 uL (“superclean”)

Ligations

  • E0240 into two backbones: pSB1C3-K823005 and pSB1C3-K823012
  • pSB1C3-K823005 SpeI/PstI/CIP: 100 ng = 0.87 uL
  • pSB1C3-K823012 SpeI/PstI/CIP: 100 ng = 1.67 uL
  • E0240 XbaI/PstI: 300 ng = 6 uL
  • 2 BO controls and 1 IO control
  • 20 mins at rt
  • Heat shock transformation and plated on LB+Cm

Objective: Test crRNA repression of GFP reporter

Dilute cultures to grow in log phase for flow

  • 1/1000 dilution into 50 mL
  • 18 samples: 3 copies each of 6 strains (labelled 2-19)
  • Samples 2-4: DH5alpha-Z1 (background)
  • grown in LB+Spec
  • Samples 5-7: DH5alpha-Z1 + pSB6A1-K608012 (reporter positive control)
  • grown in LB+Amp
  • Samples 8-10: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9 (non-target control)
  • grown in LB+Amp+Cm
  • Samples 11-13: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP1
  • grown in LB+Amp+Cm
  • Samples 14-16: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP2
  • grown in LB+Amp+Cm
  • Samples 17-19: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP3
  • grown in LB+Amp+Cm

Flow cytometry

  • Samples grown for 4.5 hrs after initial dilution
  • 1/50 final dilution of sample into PBS, placed on ice until run
  • parameters (same as previous GFP runs):
  • FSC 350V log3
  • SSC 350V log3
  • B1 350V log5
  • Trigger 10
  • 10,000 event limit (100,000 events used in the past)
  • Results
  • ~3-fold repression of reporter with crRNA_GFP1 or crRNA_GFP3
  • crRNA_GFP2 did not repress
  • non-target control repressed little or not at all
  • See document “Flow stats 7-28-14” for data details

July 29

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Minipreps

  • pSB4K5-J04450 (4 copies)
  • pSB1C3-R0040-anti-tracrRNA (4 copies)

Prep-scale digest

  • pSB4K5-J04450 (4 copies) with EcoRI/SpeI
  • pSB1C3-R0040-anti-tracrRNA (4 copies) with EcoRI/SpeI

Gel extraction and cleanup of digests

  • Cut pSB4K5 backbone from 4 copies of pSB4K5-J04450
  • 2 digests per gel well
  • 2.04 g total gel eluted into two tubes:
  • 51.4 ng/uL and 94.5 ng/uL, 30 uL each
  • Cut R0040-anti-tracrRNA insert from pSB1C3-R0040-anti-tracrRNA
  • 2 digests per gel well
  • 1.61 g total gel eluted into two tubes:
  • 99.4 ng/uL and 82.3 ng/uL, 30 uL each

Objective: Create pSB1C3-R0011-I13500

Colony PCR

  • 24 colonies of potential pSB1C3-R0011-I13500
  • 8 colonies from 7/15 ligation
  • 4x4=16 colonies from 4 different 7/21 ligation plates
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • extension time 45 sec, anneal temp 62C

Agarose gel of colony PCR results

  • colony #19 is only promising result

Analytical digest

  • pSB1C3-R0011 copies 2, 4
  • pSB1C3-I13500 copies 2, 4
  • with MfeI/NcoI
  • To test whether enzymes or original parts are the source of ligation difficulties

Agarose gel of digest results

  • Order:
  • 1. pSB1C3-R0011 #2 cut with MfeI/NcoI (expected 2 bands)
  • 2. pSB1C3-R0011 #4 cut with MfeI/NcoI (expected 2 bands)
  • 3. pSB1C3-I13500 #2 cut with MfeI/NcoI (expected 3 bands)
  • 4. pSB1C3-I13500 #4 cut with MfeI/NcoI (expected 3 bands)
  • Results:
  • R0011 both copies only show one band
  • I13500 both copies appear correct

Cultured colonies

  • Potential pSB1C3-R0011-I13500 #19 from colony PCR
  • pSB1C3-R0011 (3 copies) from original transformation
  • suspicion that our R0011 is incorrect--could be just bad copies

Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds

Culture colonies

  • pSB1C3-R0040 (4 copies)

Objective: Measurement interlab study

Plate results

  • both experimental and both backbone-only plates all had hundreds of colonies
  • insert-only controls had no colonies

Colony PCR

  • 8 colonies of potential pSB1C3-K823005-E0240
  • 8 colonies of potential pSB1C3-K823012-E0240
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • extension time 45 sec, anneal temp 62C

Agarose gel of colony PCR results

  • 1-8: pSB1C3-K823005-E0240
  • 9-16: pSB1C3-K823012-E0240
  • Results: no successes

July 30

Objective: Create pSB1C3-R0011-I13500

Minipreps

  • pSB1C3-R0011-I13500 #19
  • concentration 246.4 ng/uL
  • pSB1C3-R0011 (3 copies)
  • concentrations 187.6, 178.8, 212.8 ng/uL

Analytical digest

  • pSB1C3-R0011-I13500 #19
  • three digests in cutsmart, 10 uL final volume each
  • NcoI
  • MfeI
  • XbaI/PstI
  • pSB1C3-R0011 (3 copies)
  • with NcoI/MfeI in cutsmart, 10 uL final volume

Agarose gel of digest results

  • 1. pSB1C3-R0011-I13500 #19 with NcoI
  • 2. pSB1C3-R0011-I13500 #19 with MfeI
  • 3. pSB1C3-R0011-I13500 #19 with EcoRI/PstI
  • 4. pSB1C3-R0011 #1 with NcoI/MfeI
  • 5. pSB1C3-R0011 #2 with NcoI/MfeI
  • 6. pSB1C3-R0011 #3 with NcoI/MfeI
  • Results: hard to explain
  • R0011-I13500 not correct
  • R0011 all three copies only cut once

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Treat pSB4K5 with Antarctic Phosphatase

  • 2 copies digested on 7/29, combined into one tube with 100 uL final volume
  • 2 hrs at 37C

PCR cleanup of AP-treated pSB4K5

  • Qiagen kit
  • concentration 45.2 ng/uL in 50 uL

Ligation of pSB4K5 and R0040-anti-tracrRNA

  • 2 experimental plates with 3x and 6x molar insert
  • pSB4K5 backbone (EcoRI/SpeI/AP)100 ng = 2 uL
  • R0040-anti-tracrRNA insert (EcoRI/SpeI) 30 ng = 0.3 uL, or 60 ng = 0.6 uL
  • Backbone-only and insert-only controls
  • Transformed via heat shock and plated on LB+G418

Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds

Minipreps

  • pSB1C3-R0040 (4 copies)
  • concentrations 47.4, 80.7, 109.7, 193.5

Objective: Switch dCas9-tracrRNA into pSB1C3

Treat pSB1C3 with Antarctic Phosphatase

  • cleaned copy from 7/28 with 100 uL final volume
  • 2 hrs at 37C

PCR cleanup of AP-treated pSB1C3

  • Qiagen kit
  • concentration 104.5 ng/uL in 50 uL

Ligation of pSB1C3 and dCas9-tracrRNA

  • 2 experimental plates
  • 3x molar dCas9-tracrRNA “insert”
  • pSB1C3 100 ng = 1 uL
  • dCas9-tracrRNA 600 ng = 7.5 uL
  • 3x molar pSB1C3 “backbone”
  • pSB1C3 150 ng = 1.5 uL
  • dCas9-tracrRNA 100 ng = 1.2 uL
  • Backbone-only and insert-only controls
  • Transformed via heat shock and plated on LB+Cm

July 31

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Plate results:

  • Hundreds of colonies on both experimental plates
  • 20-30 colonies on backbone-only control
  • 0 colonies on insert-only control

Cultured colonies

  • 4 copies of pSB4K5-R0040-anti-tracrRNA

Objective: Switch dCas9-tracrRNA and dCas9-tracrRNA-crRNA into pSB1C3

Plate results:

  • Experimental plate 2 (higher pSB1C3 concentration) had 2 colonies
  • Experimental plate 1 had no colonies
  • Backbone-only control had 1 colony, insert-only control had 0

Cultured colonies

  • 2 copies of potential pSB1C3-dCas9-tracrRNA

PCRs of tracrRNA-dCas9 and tracrRNA-dCas9-crRNA

  • 8 tubes of each, with 1 uL 1 ng/uL pdCas9 template per tube
  • tracrRNA-dCas9 oligos: dCas9tracr-up and dCas9tracr-dn
  • tracrRNA-dCas9-crRNA oligos: dCas9tracr-up and pdCas9-dn
  • Q5 polymerase
  • 64C anneal temp, 2:30 extension time

Agarose gel of PCRs

  • 1-8: tracrRNA-dCas9-crRNA
  • 9-16: tracrRNA-dCas9
  • Results: all 16 lanes look good

PCR cleanup of PCRs

  • 8 tubes into 50 uL for each of two PCRs
  • Concentrations
  • tracrRNA-dCas9-crRNA: 350 ng/uL
  • tracrRNA-dCas9: 335 ng/uL

Prep-scale digest of 2 PCRs

  • tracrRNA-dCas9-crRNA and tracrRNA-dCas9
  • Each in 100 uL final volume with 2 uL each of XbaI/PstI/DpnI

PCR cleanup of digests

  • Into 50 uL final volume

Objective: Put R0040 in front of Csy4 scaffold and Repeat-seq scaffold

Prep-scale digest

  • pSB1C3-R0040 (2 copies) with 2.5 uL each of SpeI/PstI
  • 100 uL final volume each copy

PCR cleanup of digests

  • Into 50 uL final volume

Objective: Triple-transformation of R0040-anti-tracrRNA into Z1-reporter-crRNA_GFP cells

Cultured cells in morning

  • straight from frozen stocks into 2 mL LB+Cm+Amp
  • DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP1
  • DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP3

Cultured cells in evening

  • 1 mL from morning culture into 50 mL flask for overnight growth
  • To make chemically competent cells

Objective: Create pSB1C3-R0011-I13500

Cultured colonies

  • 2 copies of pSB1C3-R0011 from streak plate of original frozen stock