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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Aug
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August |
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- kivD, alsS, ilvC and ilvD
- This week we wanted to identify our positive colonies.
- Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
- Annealing temperature: 65°C
- Bands not as expected (... bp)
- kivD, alsS, ilvC, ilvD 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 kivD, alsS, ilvC and ilvD on pSB1C3
- Transformation with electrocompotetent cells
- Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
- Annealing temperature: 65°C
- Bands not as expected (... bp)
- kivD, alsS, ilvC, ilvD 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
