"
Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
(Difference between revisions)
| Line 165: | Line 165: | ||
<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>This week we aim to | + | <li>This week we aim to combine the four CDS's with the pSB1C3 backbone.</li> |
| - | <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#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" 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#RestrictionEnzymes" 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> | + | |
| - | + | ||
<ul> | <ul> | ||
</ul> | </ul> | ||
Revision as of 15:55, 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 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
