August 31st, 2010
- Slight change of plans:
- We want to have all the constructions in the same plasmid to avoid noise caused by the different plasmids, so I will change the promoter of BBa_I20260 and BBa_I20260 with the changed RBS, which original promoter is J23010, now I’ll change for our minimum Blue Promoter by PCR.
- First I have to transform BBa_I20260 with the changed RBS into DH5-α competent cells to extract this plasmid and then perform the PCR to change the promoter.
- PCR to change promoter will be done using as templates both plasmids: BBa_I20260 original and BBa_I20260 with the changed RBS.
1. Transform BBa_I20260 with the changed RBS into DH5-α competent cells, transform also pSB1T3 as control.
- Unfreeze competent cells (keep on ice).
- Add 5ul of exogenous DNA to 100ul of compentent cells.
- Incubate 20 min on ice (during this time the DNA and the cells interact to achieve transformation).
- Incubate 1 min at 42ºC (this step increases transformation efficiency, it allows membrane motitlity and close the Ca channel).
- Incubate 5 min on ice.
- Transfer the cells to a tube with 1ml of LB medium
- Incubate the cells in the LB medium for 1hr at 37ºC and shaking (during this period the cells recover their metabolic activity, replicate and synthesize the proteins encoded in the material just inserted).
- Transfer the LB medium with the cells to an eppendorf tube, centrifugue 1 min at 13000rpm.
- Discard the supernatant keeping just ~50ul of medium and resuspend the pellet in this volume.
- Plate the resuspended cells on dishes with the correspondent antibiotic. Add ~10 pearls and shake 1-2 min.
- Incubate the plates at 37ºC overnight.