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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Aug
From 2014.igem.org
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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)
- New try of the combination of 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
- 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: 80 s
- Bands as expected (2,2 kp)
- Restriction digestion with DpnI
- 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 (3,45 bp)
- alsS, ilvC, ilvD, kivD and backbone pSB1K3
- This week we tried to begin from zero again with optimized conditions and new competent cells.
Therefor we prepared new plasmids from alsS, ilvC, ilvD, kivD and the backbone pSB1K3-RFP - PCR amplification
- Primer were used as described before
- pSB1K3-RFP was used as template for backbone amplification
- Annealing temperature: 53 °C
- Bands as expected for
alsS: about 1.8 kb,
ilvC: about 1.55 kb,
ilvD: about 1.95 kb,
kivD: about 1.7 kb,
but not for pSB1K3-RFP - PCR amplification with Q5 for pSB1K3-RFP
- Primer were used as described before
- Annealing temperature: 53 °C
- Bands as expected (about 2.2 kb)
- PCR products were purified
- Gibson Assembly with kivD, alsS, ilvC and ilvD on pSB1K3
- Restriction digestion with DpnI
- Aberation from protocol: Incubation for about 10 hours.
- 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 pSB1K3-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
