"
Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
(Difference between revisions)
| Line 56: | Line 56: | ||
<li>Optimization of PCR conditions for coding sequence amplification</li> | <li>Optimization of PCR conditions for coding sequence amplification</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Combinations of primers and templates as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li> |
| - | <li> | + | <li>Annealing temperature gradients from 50°C to 58°C were tried</li> |
| - | <li> | + | <li>Product amount was increased by lower annealing temperatures</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| Line 88: | Line 88: | ||
<li>Optimization of PCR conditions for coding sequence amplification</li> | <li>Optimization of PCR conditions for coding sequence amplification</li> | ||
<ul> | <ul> | ||
| - | <li> | + | <li>Combinations of primers and templates as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li> |
| - | <li> | + | <li>Annealing temperature gradients from 50°C to 58°C were tried</li> |
| - | <li> | + | <li>Product amount was increased by lower annealing temperatures</li> |
<ul> | <ul> | ||
| - | <li>54°C | + | <li>For the annealing temperature we identified 54°C as optimal.</li> |
| - | <li>90 seconds | + | <li>For the elongation time 90 seconds worked better than 60 seconds.</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
| Line 122: | Line 122: | ||
<li><b><i>kivD</i>, <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i></b></li> | <li><b><i>kivD</i>, <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i></b></li> | ||
<ul> | <ul> | ||
| - | <li>With optimized conditions the | + | <li>With the optimized conditions the amplifications were tried again.</li> |
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></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></li> |
<li>PCR products were extracted out of the gel.</li> | <li>PCR products were extracted out of the gel.</li> | ||
| - | <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> | </ul> | ||
<li><b>Backbone of pSB1C3</b></li> | <li><b>Backbone of pSB1C3</b></li> | ||
<ul> | <ul> | ||
| - | <li>We aim to amplify | + | <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>)</li> | ||
<ul> | <ul> | ||
| - | <li>Elongation time: | + | <li>Elongation time: 90</li> |
| - | <li>Bands | + | <li>Bands not as expected (2,2 kb)</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> | ||
Revision as of 15:36, 30 August 2014
 |
July |
 |
- kivD, alsS, ilvC, ilvD and backbone of 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
- kivD, alsS, ilvC, ilvD and backbone of 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.
- kivD, alsS, ilvC, ilvD
- 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
- Backbone of pSB1C3
- We aim to amplify it with Q5 polymerase
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3)
- Elongation time: 90
- Bands not as expected (2,2 kb)
- PCR purification of backbone
- kivD, alsS, ilvC, ilvD
- This week we aim to prove if the constructs were right
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- Gibson Assembly with kivD, alsS, ilvC and ilvD on pSB1C3
- Transformation with electrocompotetent cells
