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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Aug
From 2014.igem.org
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 1 08/04 - 08/10</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
<ul> | <ul> | ||
| - | <li><b> | + | <li><b>psB1C3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i></b></li> |
<ul> | <ul> | ||
| - | <li>This week we wanted to identify | + | <li>This week we wanted to identify positive colonies.</li> |
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
| - | <li>Bands not as expected ( | + | <li>Bands not as expected (~ 3450 bp) </li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| + | <li>New try of the combination of the four CDS's with the pSB1C3 backbone.</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> and pSB1C3</li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>DpnI</i></a></li> | ||
| + | <li>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompetent</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank"> chemocompetent</a> cells</li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65 °C</li> | ||
| + | <li>Bands not as expected (~ 3450 bp) </li> | ||
| + | </ul> | ||
| + | <ul> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<div class="tab" id="Week2"> | <div class="tab" id="Week2"> | ||
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<h6><a style="font-size:24px" href="#">Week 2 08/11 - 08/17</a></h6> | <h6><a style="font-size:24px" href="#">Week 2 08/11 - 08/17</a></h6> | ||
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
<ul> | <ul> | ||
| - | <li><b><i> | + | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone of pSB1C3</b></li> |
<ul> | <ul> | ||
<li>We started again back by zero to solve our problems.</li> | <li>We started again back by zero to solve our problems.</li> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described before)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described before)</li> | ||
<ul> | <ul> | ||
| - | <li>We used pSB1C3-RFP instead of pSB1C3-CFP as template for backbone amplification</li> | + | <li>We used pSB1C3-RFP (<a href="http://parts.igem.org/Part:BBa_J04450" target="_blank">J04450</a>) instead of pSB1C3-CFP as template for backbone amplification</li> |
| - | <li> | + | <li>Annealing temperature: 65 °C</li> |
| - | <li>Bands | + | <li>Bands as expected (~ 2200 bp)</li> |
</ul> | </ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes" target="_blank"><i>DpnI</i></a></li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes" target="_blank"><i>DpnI</i></a></li> | ||
| - | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i> and <i>kivD</i> on pSB1C3</li> | |
| - | + | ||
| - | + | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i> | + | |
<li>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompotetent cells</a></li> | <li>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompotetent cells</a></li> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
| - | <li>Bands not as expected ( | + | <li>Bands not as expected (~ 3450 bp)</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 3 08/18 - 08/24</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
<ul> | <ul> | ||
| - | <li><b><i> | + | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1K3</b></li> |
<ul> | <ul> | ||
| - | <li>This week we tried to begin from zero again with optimized conditions and new competent cells.</li> | + | <li>This week we tried to begin from zero again with optimized conditions and new competent cells. <br>Therefor we prepared new plasmids from <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and the backbone pSB1K3-RFP</li> |
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a></li> |
<ul> | <ul> | ||
<li>Primer were used as described before</li> | <li>Primer were used as described before</li> | ||
<li>pSB1K3-RFP was used as template for backbone amplification</li> | <li>pSB1K3-RFP was used as template for backbone amplification</li> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 53 °C</li> |
| - | <li>Bands | + | <li>Bands as expected for <br><i>alsS</i>: about 1800 bp, <br><i>ilvC</i>: about 1550 bp, <br><i>ilvD</i>: about 1950 bp, <br><i>kivD</i>: about 1700 bp, <br>but not for pSB1K3-RFP</li> |
</ul> | </ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes# | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a></li> with Q5 for pSB1K3-RFP |
| + | <ul> | ||
| + | <li>Primer were used as described before</li> | ||
| + | <li>Annealing temperature: 53 °C</li> | ||
| + | <li>Bands as expected (~ 2200 bp)</li> | ||
| + | </ul> | ||
| + | |||
| + | <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li> | ||
| + | |||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> on pSB1K3</li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>DpnI</i></a></li> | ||
<ul> | <ul> | ||
<li>Aberation from protocol: Incubation for about 10 hours.</li> | <li>Aberation from protocol: Incubation for about 10 hours.</li> | ||
| - | + | </ul><li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | |
| - | </ul> | + | |
| - | + | ||
| - | + | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
| - | <li>Bands as expected ( | + | <li>Bands as expected (~ 3600 bp)</li> |
</ul> | </ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
| - | <li>Bands as expected ( | + | <li>Bands as expected (~ 3300 bp)</li> |
</ul> | </ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of | + | <li>Liquid cultures for a restriction digest were prepared.</li> |
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes# | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1K3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i></li> |
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>NotI</i></a></li> | ||
<ul> | <ul> | ||
| - | <li>Bands as expected (backbone: | + | <li>Bands as expected (backbone:~ 2200 bp and insert: ~ 7000 bp)</li> |
</ul> | </ul> | ||
| - | |||
</ul> | </ul> | ||
</ul> | </ul> | ||
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| - | <div id="text"> | + | <div id="text" style="text-align:left"> |
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<div class="hide"> | <div class="hide"> | ||
| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 4 08/25 - 08/31</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| - | </div> | + | |
| + | <ul> | ||
| + | <li><b><i>adhA</i></b></li> | ||
| + | <ul> | ||
| + | <li>This week the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis" target="_blank"> <i>Lactococcus lactis</i></a> arrived. We tried to amplify it's <i>adhA</i>.</li> | ||
| + | <ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAisolation" target="_blank">DNA isolation</a> of <i>Lactococcus lactis</i></li> | ||
| + | </ul> | ||
| + | |||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the <i>adhA</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-pBS1C3-adhA" target="_blank">rev-pBS1C3-adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-pSB1C3_adhA" target="_blank">fw-pSB1C3_adhA</a>) and the Backbone out of psB1K3-<i>RFP</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-adhA_pSB1C3" target="_blank">rev-adhA_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-adhA-pSB1C3" target="_blank">fw-adhA-pSB1C3</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 54 °C</li> | ||
| + | <li>Bands as expected (~1200 bp for the <i>adhA</i> and ~ 2200 bp for the Backbone)</li> | ||
| + | </ul> | ||
| + | |||
| + | </ul> | ||
| + | <ul> | ||
| + | <li>PCR products were extracted out of the gel and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a>.</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>adhA</i> and pSB1K3</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Dpn1</i></a> <ul/> | ||
| + | |||
| + | |||
| + | |||
| + | </ul> | ||
| + | |||
| + | </ul> | ||
| + | <ul> | ||
| + | <li> Transformation <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank"> with chemocompetent cells</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-pBS1C3-adhA target="_blank">rev-pBS1C3-adhArev-pBS1C3-adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-pSB1C3_adhA" target="_blank">fw-pSB1C3_adhA</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65°C</li> | ||
| + | <li>Bands as expected (~ 1200 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <li><b><i>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i> with T7-Promotor</b></li> | ||
| + | <ul> | ||
| + | <li> BioBrick Assembly: | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li> | ||
| + | <ul> | ||
| + | <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li>pSB1A2-T7</li> | ||
| + | </ul> | ||
| + | <li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
| + | <ul> | ||
| + | <li><i>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i></li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | |||
| + | </ul> | ||
| + | |||
| + | |||
| + | </li> | ||
| + | </ul><ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-pSB1C3-alsS" target="_blank">fw-pSB1C3-alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv-ilvC-alsS" target="_blank">rv-ilvC-alsS</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 65 °C</li> | ||
| + | <li>Bands as expected (~ 1800 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li>Liquid cultures for a restriction digest were prepared.</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <br> | ||
| + | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 16:49, 16 October 2014
 |
August |
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- psB1C3-alsS-ilvC-ilvD
- This week we wanted to identify positive colonies.
- Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
- Annealing temperature: 65 °C
- Bands not as expected (~ 3450 bp)
- New try of the combination of the four CDS's with the pSB1C3 backbone.
- Gibson Assembly with kivD, alsS, ilvC and ilvD and pSB1C3
- Restriction digestion with DpnI
- Transformation with electrocompetent and chemocompetent cells
- Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
- Annealing temperature: 65 °C
- Bands not as expected (~ 3450 bp)
- alsS, ilvC, ilvD, kivD and backbone of pSB1C3
- We started again back by zero to solve our problems.
- PCR amplification (as described before)
- We used pSB1C3-RFP (J04450) instead of pSB1C3-CFP as template for backbone amplification
- Annealing temperature: 65 °C
- Bands as expected (~ 2200 bp)
- Restriction digestion with DpnI
- Gibson Assembly with alsS, ilvC, ilvD and kivD on pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
- Annealing temperature: 65 °C
- Bands not as expected (~ 3450 bp)
- alsS, ilvC, ilvD, kivD and backbone pSB1K3
- This week we tried to begin from zero again with optimized conditions and new competent cells.
Therefor we prepared new plasmids from alsS, ilvC, ilvD, kivD and the backbone pSB1K3-RFP - PCR amplification
- Primer were used as described before
- pSB1K3-RFP was used as template for backbone amplification
- Annealing temperature: 53 °C
- Bands as expected for
alsS: about 1800 bp,
ilvC: about 1550 bp,
ilvD: about 1950 bp,
kivD: about 1700 bp,
but not for pSB1K3-RFP - PCR amplification with Q5 for pSB1K3-RFP
- Primer were used as described before
- Annealing temperature: 53 °C
- Bands as expected (~ 2200 bp)
- PCR products were purified
- Gibson Assembly with kivD, alsS, ilvC and ilvD on pSB1K3
- Restriction digestion with DpnI
- Aberation from protocol: Incubation for about 10 hours.
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvC_ilvD, rv_pSB1C3_kivD)
- Annealing temperature: 65 °C
- Bands as expected (~ 3600 bp)
- Colony PCR (fw_alsS_ilvC, rv_ilvD_ilvC)
- Annealing temperature: 65 °C
- Bands as expected (~ 3300 bp)
- Liquid cultures for a restriction digest were prepared.
- Plasmid isolation of pSB1K3-alsS-ilvC-ilvD-kivD
- Restriction digestion with NotI
- Bands as expected (backbone:~ 2200 bp and insert: ~ 7000 bp)
- adhA
- This week the Lactococcus lactis arrived. We tried to amplify it's adhA.
- DNA isolation of Lactococcus lactis
- PCR amplification of the adhA (rev-pBS1C3-adhA, fw-pSB1C3_adhA) and the Backbone out of psB1K3-RFP (rev-adhA_pSB1C3, fw-adhA-pSB1C3)
- Annealing temperature: 54 °C
- Bands as expected (~1200 bp for the adhA and ~ 2200 bp for the Backbone)
- PCR products were extracted out of the gel and purified.
- Gibson Assembly with adhA and pSB1K3
- Transformation with chemocompetent cells
- Colony PCR (rev-pBS1C3-adhArev-pBS1C3-adhA, fw-pSB1C3_adhA)
- Annealing temperature: 65°C
- Bands as expected (~ 1200 bp)
- BioBrick Assembly:
- BioBrick Assembly (Suffix)
- Transformation with electrocompotetent cells
- Colony PCR (fw-pSB1C3-alsS, rv-ilvC-alsS)
- Annealing temperature: 65 °C
- Bands as expected (~ 1800 bp)
- Liquid cultures for a restriction digest were prepared.
