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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
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July |
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- kivD, alsS, ilvC, ilvD and backbone of pSB1C3
- We tried to redo the amplification of last week.
- 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
- kivD, alsS, ilvC, ilvD and backbone of pSB1C3
- We tried to redo the amplification of last week.
- 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
- kivD, alsS, ilvC, ilvD
- With optimized conditions the amplification should give results this week
- PCR amplification as described before
- PCR products were extracted out of the gel.
- Plasmid purfication of the constructs
- Backbone of pSB1C3
- We aim to amplify the backbone with Q5 polymerase
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3)
- Elongation time: ...
- Bands (not) as expected (... bp)
- PCR purification of backbone
- kivD, alsS, ilvC, ilvD
- This week we aim to prove if the constructs were right
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- Gibson Assembly with kivD, alsS, ilvC and ilvD on pSB1C3
- Transformation with electrocompotetent cells
