September 28th, 2010
1. Inactivate ligation enzyme for 20 min at 65ºC.
2. Transform the following ligations:
- BBa_J61002 plasmid (pSB1A2) containing promoter J23101 (SpeI-PstI restriction) + Δ RBS-GFP E0040 (10 ul)
- Religation of pSB3K3 + J23101 (SpeI restriction) (10 ul)
- Religation of pSB4A5 (p30) + Minimmum Blue Promoter (10 ul)
into DH5-α competent cells, transform also pSB4A5 (p30) (5 ul DNA) as control.
- Unfreeze competent cells (keep on ice).
- Add exogenous DNA to 50ul 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.