Team:Aachen/Wetlab/Protocol13

Transformation of chemical competent E. coli cells:

Procedure:


  1. Thaw TSS cells on ice.

  2. Add DNA, pipette gently to mix (1 ul of prepped plasmid is more than enough).>

  3. Let sit for 30 minutes on ice.

  4. Incubate cells for 30-45 seconds at 42 ℃.

  5. Incubate cells on ice for 2 min.

  6. Add 1 mL SOC (2XYT and LB are also suitable) at room temp

  7. Incubate for 45 minutes - 1 hour at 37 ℃ on shaker.

  8. Spread 100-300 ul onto a plate made with appropriate antibiotic.

  9. Grow overnight at 37 ℃.

Notes:


  • Save the rest of the transformants in liquid culture at 4 ℃. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.

  • With SOC-medium you have a 3-folded better transformation efficiency of E. coli cells.>

  • If you are adding small volumes of plasmid or transormation DNA (~1 ul), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.

  • If you are in a rush, you can shorten incubation time on ice to 5-10 min.(step 3)

  • According to the original TSS paper, heat shock step is completely optional and may actually reduce transformation efficiency.With DH5a Z1 and pUC19 a heat shock at 42 ℃ for 30 sec improved transformation efficiency 10-fold.