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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
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<ul> | <ul> | ||
| - | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone | + | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1C3</b></li> |
<ul> | <ul> | ||
<li>We tried to redo the amplification of last week.</li> | <li>We tried to redo the amplification of last week.</li> | ||
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<div class="content" style="margin-right:10%; margin-left:10%"> | <div class="content" style="margin-right:10%; margin-left:10%"> | ||
<ul> | <ul> | ||
| - | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone | + | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1C3</b></li> |
<ul> | <ul> | ||
<li>We tried to redo the amplification of last week.</li> | <li>We tried to redo the amplification of last week.</li> | ||
| Line 127: | Line 127: | ||
<li>PCR products were extracted out of the gel.</li> | <li>PCR products were extracted out of the gel.</li> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purfication</a> of the gel slices </li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purfication</a> of the gel slices </li> | ||
| - | + | <li>This worked well for <i>ilvC</i>, but not for <i>alsS</i>, <i>ilvD</i> and <i>kivD</i>.</li> | |
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> with the optimized conditions and the primer combinations as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a> for <i>alsS</i>, <i>ilvD</i> and <i>kivD</i></li> | ||
| + | <li>PCR products were extracted out of the gel.</li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purfication</a> of the gel slices </li> | ||
| + | </ul> | ||
</ul> | </ul> | ||
| - | <li><b>Backbone | + | <li><b>Backbone pSB1C3</b></li> |
<ul> | <ul> | ||
<li>We aim to amplify it with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#Polymerases" target="_blank">Q5 polymerase</a></li> | <li>We aim to amplify it with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#Polymerases" target="_blank">Q5 polymerase</a></li> | ||
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li> | + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>) (Elongationtime: 60 sec)</li> |
| + | <ul> | ||
| + | <li>Annealing temperature: 54 °C</li> | ||
| + | <li>Bands not as expected (~ 2.200 bp)</li> | ||
| + | </ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>) (Elongationtime: 80 sec)</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Annealing temperature: 54 °C</li> |
| - | <li>Bands | + | <li>Bands as expected (~ 2.200 bp)</li> |
</ul> | </ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbone</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbone</li> | ||
| Line 168: | Line 177: | ||
<ul> | <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#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# | + | <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 chemocompetent cells</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> |
<ul> | <ul> | ||
</ul> | </ul> | ||
Latest revision as of 16:00, 16 October 2014
 |
July |
 |
- alsS, ilvC, ilvD, kivD and backbone pSB1C3
- We tried to redo the amplification of last week.
- Optimization of PCR conditions for coding sequence amplification
- Combinations of primers and templates as described before
- Annealing temperature gradients from 50°C to 58°C were tried
- Product amount was increased by lower annealing temperatures, but still not good enough for Gibson
- alsS, ilvC, ilvD, kivD and backbone pSB1C3
- We tried to redo the amplification of last week.
- Optimization of PCR conditions for coding sequence amplification
- Combinations of primers and templates as described before
- Annealing temperature gradients from 50°C to 58°C were tried
- Product amount was increased by lower annealing temperatures
- For the annealing temperature we identified 54°C as optimal.
- For the elongation time 90 seconds worked better than 60 seconds.
- alsS, ilvC, ilvD, kivD
- With the optimized conditions the amplifications were tried again.
- PCR amplification with the optimized conditions and the primer combinations as described before
- PCR products were extracted out of the gel.
- Purfication of the gel slices
- This worked well for ilvC, but not for alsS, ilvD and kivD.
- PCR amplification with the optimized conditions and the primer combinations as described before for alsS, ilvD and kivD
- PCR products were extracted out of the gel.
- Purfication of the gel slices
- Backbone pSB1C3
- We aim to amplify it with Q5 polymerase
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3) (Elongationtime: 60 sec)
- Annealing temperature: 54 °C
- Bands not as expected (~ 2.200 bp)
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3) (Elongationtime: 80 sec)
- Annealing temperature: 54 °C
- Bands as expected (~ 2.200 bp)
- PCR purification of backbone
- alsS, ilvC, ilvD, kivD
- This week we aim to combine 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
