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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)
- pSB1C3-alsS-ilvC-ilvD-kivD
- Amplification of all five parts was repeated with Q5 polymerase from NEB
- PCR conditions were set as used before
- pSB1K3-RFP was used as template for pSB1K3 backbone amplification (kanamycin resistance was choosen to have another selection marker)
- PCR products were purified using Wizard® SV Gel an PCR Clean-Up System (Promega)
- DpnI digest of template molecules in purified PCR products of the backbone
- 3µL (30 units) of DpnI (Fermentas) were used for 30 µL plasmid solution (about 3 µg of DNA)
- Incubation at 37°C for about ten hours
- Gibson Assembly with all four coding sequences (alsS, ilvC, ilvD, kivD) and pSB1C3
- Transformation of electrocompetent E. coli KRX cells
- Colony PCR (fw_ilvC_ilvD, rv_pSB1C3_kivD)
- Annealing temperature was set to 65°C to achieve strict conditions
- Agarose gel electrophoresis showed products of expected size (about 3.6kb)
- Colony PCR (fw_alsS_ilvC, rv_ilvD_ilvC)
- Annealing temperature was set to 65°C to achieve strict conditions
- Agarose gel electrophoresis showed products of expected size (about 3.3kb)
- Positive clones were used to start liquid cultures
- Plasmid isolation of pSB1K3-alsS-ilvC-ilvD-kivD
- NotI digestion of isolated plasmids
- Components (10µL total volume):
- 0.3µL NotI (Fermentas)
- 1µL 10 fold Organge buffer (Fermentas)
- 3µL plasmid solution (< µg of DNA)
- 5.7µL water
- Incubation at 37°C for one hour
- Agarose gel electrophoresis showed expected fragment sizes:
- 2.2kb - backbone
- 6.7kb - insert
- Glycerin stocks for positive clones created
- Sanger sequencing using VF and VR as sequencing primers
