September 8th, 2010
1. Inactivate ligation enzyme for 20 min at 65ºC.
2. Transform the ligation of BBa_I20260 Δ RBS (using the 20 ul of the ligation reaction) 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.