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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
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July |
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- alsS, ilvC, ilvD, kivD 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, but still not good enough for Gibson
- alsS, ilvC, ilvD, kivD 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
- For the annealing temperature we identified 54°C as optimal.
- For the elongation time 90 seconds worked better than 60 seconds.
- alsS, ilvC, ilvD, kivD
- With the optimized conditions the amplifications were tried again.
- PCR amplification with the optimized conditions and the primer combinations as described before
- PCR products were extracted out of the gel.
- Purfication of the gel slices
- This worked well for ilvC, but not for alsS, ilvD and kivD.
- PCR amplification with the optimized conditions and the primer combinations as described before for alsS, ilvD and kivD
- PCR products were extracted out of the gel.
- Purfication of the gel slices
- Backbone of pSB1C3
- We aim to amplify it with Q5 polymerase
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3)
- Elongation time: 90 s
- Bands not as expected (2,2 kb)
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3)
- Elongation time: 80 s
- Bands as expected (2,2 kb)
- PCR purification of backbone
- alsS, ilvC, ilvD, kivD
- This week we aim to combine 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
