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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
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July |
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- pSB1C3-alsS-ilvC-ilvD-kivD
- Optimization of PCR conditions for coding sequence amplification
- combinations of primers and templates as described before
- annealing temperature gradients from 50°C to 58°C were tried
- product amount was increased by lower annealing temperatures
- pSB1C3-alsS-ilvC-ilvD-kivD
- Optimization of PCR conditions for coding sequence amplification
- combinations of primers and templates as described before
- annealing temperature gradients from 50°C to 58°C were tried
- product amount was increased by lower annealing temperatures
- 54°C was identified as optimal annealing temperature
- 90 seconds were identified as optimal elongation time
- pSB1C3-alsS-ilvC-ilvD-kivD
- Amplification of coding sequences was repeated using optimized conditions
- Gel extraction of PCR products using GeneJET Gel Extraction Kit from ThermoScientific
- PCR product prufication was carried out using GeneJET PCR Purification Kit from ThermoScientific
- pSB1C3 backbone was amplified using Q5 polymerase from NEB
- PCR products were analyzed by agarose gel electrophoresis
- PCR product purification by Wizard SV Gel and PCR Clean-Up System (Promega)
- pSB1C3-alsS-ilvC-ilvD-kivD
- DpnI digest of template molecules in all purified PCR samples
- 1µL (10 units) of enzyme were used for 30 µL plasmid solution (about 3 µg of DNA)
- Incubation at 37°C for three hours
- Gibson Assembly with all four coding sequences (alsS, ilvC, ilvD, kivD) and pSB1C3
- Transformation of electrocompetent E. coli KRX cells
