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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
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| - | <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 tried to redo the amplification of last week.</li> | <li>We tried to redo the amplification of last week.</li> | ||
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<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 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"> | <div class="content"> | ||
<ul> | <ul> | ||
| - | <li><b><i> | + | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i></b></li> |
<ul> | <ul> | ||
<li>With the optimized conditions the amplifications were tried again.</li> | <li>With the optimized conditions the amplifications were tried again.</li> | ||
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<ul> | <ul> | ||
| - | <li><b><i> | + | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i></b></li> |
<ul> | <ul> | ||
<li>This week we aim to combine the four CDS's with the pSB1C3 backbone.</li> | <li>This week we aim to combine the four CDS's with the pSB1C3 backbone.</li> | ||
Revision as of 16:17, 30 August 2014
 |
July |
 |
- alsS, ilvC, ilvD, kivD 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
- alsS, ilvC, ilvD, kivD 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.
- 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
- 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
- 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
