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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Aug
From 2014.igem.org
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<h6><a style="font-size:24px" href="#">Week 1 08/04 - 08/10</a></h6> | <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>psB1C3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i></b></li> | <li><b>psB1C3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i></b></li> | ||
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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>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone of pSB1C3</b></li> | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone of pSB1C3</b></li> | ||
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<h6><a style="font-size:24px" href="#">Week 3 08/18 - 08/24</a></h6> | <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>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1C3</b></li> | <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1C3</b></li> | ||
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<h6><a style="font-size:24px" href="#">Week 4 08/25 - 08/31</a></h6> | <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> | </div> | ||
</div> | </div> | ||
Revision as of 10:36, 31 August 2014
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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 (3,45 kb)
- 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 instead of pSB1C3-CFP as template for backbone amplification
- Elongation time: ...
- Bands (not) as expected (... bp)
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- 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 (... bp)
- alsS, ilvC, ilvD, kivD and backbone pSB1C3
- This week we tried to begin from zero again with optimized conditions and new competent cells.
- PCR amplification)
- Primer were used as described before
- pSB1K3-RFP was used as template for backbone amplification
- Annealing temperature: ...
- Bands (not) as expected (... bp)
- Restriction digestion with DpnI
- Aberation from protocol: Incubation for about 10 hours.
- Bands (not) as expected (... bp)
- Gibson Assembly with kivD, alsS, ilvC and ilvD on pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (fw_ilvC_ilvD, rv_pSB1C3_kivD)
- Annealing temperature: 65 °C
- Bands as expected (3,6 kb)
- Colony PCR (fw_alsS_ilvC, rv_ilvD_ilvC)
- Annealing temperature: 65 °C
- Bands as expected (3,3 kb)
- Plasmid isolation of pSB1C3-alsS-ilvC-ilvD-kivD
- Restriction digestion with NotI
- Bands as expected (backbone: 2,2 kb and insert: 6,7 kb)
- A glycerin stock was created for positive clones
