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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Aug
From 2014.igem.org
(Difference between revisions)
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
| - | <li>Bands not as expected 3,45/li> | + | <li>Bands not as expected (3,45 kb) </li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
<li>Bands not as expected (... bp)</li> | <li>Bands not as expected (... bp)</li> | ||
</ul> | </ul> | ||
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
<li>Bands as expected (3,6 kb)</li> | <li>Bands as expected (3,6 kb)</li> | ||
</ul> | </ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li> | ||
<ul> | <ul> | ||
| - | <li>Annealing temperature: | + | <li>Annealing temperature: 65 °C</li> |
<li>Bands as expected (3,3 kb)</li> | <li>Bands as expected (3,3 kb)</li> | ||
</ul> | </ul> | ||
Revision as of 16:43, 30 August 2014
 |
August |
 |
- 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
