Team:Waterloo/Lab Book

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       <h2>CRISPRi</h2>
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       <img src="https://static.igem.org/mediawiki/2014/e/e2/CRISPRi_1.png"></a><br>
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    <strong>June 6, 2014</strong><br>Digested pSB1C3 backbone with EcoRI and PstI and gel purified<br>
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      <img src="https://static.igem.org/mediawiki/2014/5/53/CRISPRi_2.png"></a><br>
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    <strong>June 9, 2014</strong><br>Received synthesized sgRNA 1 and sgRNA 2 constructs from BioBasic and transformed the DNA into E. coli DH5α <br>
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      <img src="https://static.igem.org/mediawiki/2014/d/d6/CRISPR_3.png"></a><br>
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    <strong>June 11, 2014</strong><br>Inoculated sgRNA 1 and 2 colonies from the transformation into over night liquid culture for frozen stocks and miniprep<br>
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      <img src="https://static.igem.org/mediawiki/2014/f/f9/CRISPR_4.png"></a><br>
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    <strong>June 18, 2014</strong><br>Digested sgRNA 1 and 2 constructs with EcoRI and PstI, then gel purified the inserts for cloning into pSB1A3
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       <img src="https://static.igem.org/mediawiki/2014/1/1d/CRISPR_5.png"></a><br>
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Also digested sgRNA 1 with EcoRI and SpeI, and sgRNA 2 with XbaI and PstI for modified 3A assembly into pSB1A3<br>
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      <img src="https://static.igem.org/mediawiki/2014/5/5c/CRISPRi_6.png"></a><br>
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    <strong>June 19, 2014</strong><br>Digested pSB1A3 linearized backbone with EcoRI and PstI, then column purified. <br>Ligated pSB1A3 with the sgRNA 1 and 2 (E/P) inserts individually and with both sgRNA 1 (E/S) and sgRNA 2 (X/P) together <br>Ligated pSB1C3 backbone with sgRNA 1 (E/P) and sgRNA 2 (E/P) individually for BioBricks<br>
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      <img src="https://static.igem.org/mediawiki/2014/8/82/CIRSPRi_7.png"></a><br>
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    <strong>June 20, 2014</strong><br>Transformed all ligations into E. coli DH5α<br>
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      <img src="https://static.igem.org/mediawiki/2014/0/0b/CRISPRi_8.png"></a><br>
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    <strong>June 21, 2014</strong><br>Streak purified 4 colonies from each ligation<br>
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      <img src="https://static.igem.org/mediawiki/2014/3/37/CRISPRi_9.png"></a><br>
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    <strong>June 22, 2014</strong><br>Inoculated 4 colonies of each ligation into overnight liquid culture for miniprep (patched colonies onto agar plates before inoculating<br>
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    <strong>June 23, 2014</strong><br>Miniprepped and did a diagnostic digest to see which clones contained the appropriate inserts:
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-sgRNA 1 in pSB1A3                          1 successful<br>
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-sgRNA 2 in pSB1A3                          3 successful<br>
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-sgRNA 1 and sgRNA 2 in pSB1A3       0 successful<br>
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-sgRNA 1 in pSB1C3                          1 successful<br>
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-sgRNA 2 in pSB1C3                          2 successful<br>
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    <strong>June 24, 2014</strong><br>Streak purified 4 more colonies from the sgRNA 1 and sgRNA 2 in pSB1A3 ligation to screen for successful clones<br>
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    <strong>June 25, 2014</strong><br>Inoculated the 4 clones of sgRNA 1 and sgRNA 2 in pSB1A3 for miniprep (patched colonies onto agar plates before inoculating)<br>
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    <strong>June 26, 2014</strong><br>Miniprepped and did a test digest to screen for clones with the correct insert size: 0 successful<br>
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Decided to wait for the sgRNA 1 in pSB1A3 to be sequence confirmed and then clone in sgRNA 2 instead of doing modified 3A assembly<br>
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    <strong>July 3, 2014</strong><br>Inoculated successful clones from patches into over night liquid culture to miniprep and send for sequencing<br>
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    <strong>July 4, 2014</strong><br>Miniprepped and sent clones for sequencing<br>
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    <strong>July 9, 2014</strong><br>Received synthesized dCas9 cassette from BioBasic and transformed the DNA into E. coli DH5α<br>
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    <strong>July 10, 2014</strong><br>Digested the dCas9 construct with EcoRI and PstI then gel purified the insert<br>
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Digested more pSB1A3 and pSB1C3 with EcoRI and PstI and gel purified the backbone.<br>
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Ligated the dCas9 cassette (E/P) into pSB1A3 (E/P) and pSB1C3 (E/P)<br>
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    <strong>July 11, 2014</strong><br>Transformed all ligations into E. coli DH5α<br>
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    <strong>July 12, 2014</strong><br>Streak purified 8 colonies from each ligation<br>
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    <strong>July 13, 2014</strong><br>Inoculated the colonies into over night liquid cultures for miniprep (patched colonies onto agar plates before inoculating)<br>
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    <strong>July 14, 2014</strong><br>Miniprepped and did a diagnostic digest to see which clones contained the appropriate inserts:<br>
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- dCas9 cassette in pSB1A3      0 successful<br>
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- dCas9 cassette in pSB1C3      2 successful<br>
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Ran digests of sgRNA for gel extraction<br>
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    <strong>July 15, 2014</strong><br>Streak purified 8 colonies from dCas9 pSB1A3<br>
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Gel extracted sgRNA<br>
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Restriction digest and gel extraction of dsRed-ermM-oriV, ermM-oriV, pSB1C3, pSB1A3<br>
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    <strong>July 16, 2014</strong><br>Inoculated and patched 8 colonies of dCas9 pSB1A3<br>
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Miniprepped sequence-confirmed sgRNA strains, pSB1C3, pSB1A3, ermM-oriV, dsRed-ermM-oriV<br>
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Digested, column purified, and ligated<br>
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    <strong>July 17, 2014</strong><br>Miniprep and restriction digest of 8 colonies of dCas9 pSB1A3                                  0 successful<br>
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Transformed ligations of sgRNA, pSB1C3, pSB1A3, and ermM-oriV<br>
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    <strong>July 18, 2014</strong><br>Streak purified 4 colonies for each ligation<br>
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    <strong>July 19, 2014</strong><br>Miniprepped and digested:<br>
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sgRNA, ermM-oriV, sgRNA-ermM-oriV, dCas9-ermM-oriV<br>
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sgRNA1+sgRNA2+pSB1A3                        2 successful<br>
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sgRNA1+pSB1C3                                      4 successful<br>
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ermM-oriV-pSB1A3                                  1 successful<br>
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sgRNA1-ermM-oriV-pSB1A3                      0 successful<br>
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sgRNA2-ermM-oriV-pSB1A3                      1 successful<br>
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dCas9-ermM-oriV-pSB1A3                        2 successful<br>
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    <strong>July 22, 2014</strong><br>ermM-oriV PCR<br>
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    <strong>July 24, 2014</strong><br>ermM-oriV restriction digest and ligation in pSB1A3<br>
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Miniprep and digest: spc construct-pUC57, dCas9 cassette<br>
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dsRed-ermM-oriV in pUC57 digest and gel extraction<br>
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    <strong>July 25, 2014</strong><br>dsRed-ermM-oriV digest and transformation into DC10B<br>
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Transformation: ermM-oriV-pSB1A3              4 colonies<br>
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    <strong>July 27, 2014</strong><br>Miniprep and digest of:<br>
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ermM-oriV-pSB1A3                                        3 successful<br>
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dsRed-ermM-oriV-pUC57-DC10B                    3 successful<br>
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    <strong>July X, 2014</strong><br>TEXT<br>
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    <strong>July X, 2014</strong><br>TEXT<br>
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    <strong>July X, 2014</strong><br>TEXT<br>
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    <strong>July X, 2014</strong><br>TEXT<br>
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    <strong>July X, 2014</strong><br>TEXT<br>
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    </div>
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      <h2>RNAi</h2>
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       <h2>YFP</h2>
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       <img src="https://static.igem.org/mediawiki/2014/1/1d/YFP_1.png"></a><br>
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      <img src="https://static.igem.org/mediawiki/2014/5/59/YFP_2.png"></a><br>
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       <h2>CONJUGATION</h2>
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       <img src="https://static.igem.org/mediawiki/2014/2/28/Conjugation_1.png"></a><br>
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      <img src="https://static.igem.org/mediawiki/2014/0/0a/Conjugation_2.png"></a><br>
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       <h2>OTHER</h2>
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       <img src="https://static.igem.org/mediawiki/2014/8/8e/Other_1.png"></a><br>
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    asdfasdfasdfasdfasgasdgasdg
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      <img src="https://static.igem.org/mediawiki/2014/7/7d/Other_2.png"></a><br>
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     </div>
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   </div>
   </div>

Latest revision as of 03:28, 18 October 2014

Lab Book