Preparing chemically competent cells

From OpenWetWare

Revision as of 11:38, 27 July 2005 by ClarkeS (Talk | contribs)
Jump to: navigation, search


  1. Grow a 5 - 25 mL culture to an OD600 of 0.2 - 0.5.
  2. Centrifuge for 10 minutes at 3000 rpm and 4oC.
  3. Remove supernatant and replace with 10% original volume TSS.
  4. Make 100 uL aliquots and store at − 80oC.


  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    • Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
  3. Let sit for 30 minutes on ice.
    • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 30 seconds at 42oC.
    • Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency.
  5. Add 1 mL SOC (2XYT and LB are also suitable) at room temp.
  6. Incubate for 1 hour at 37oC.
    • Note: Can also save some time here by reducing incubation to ~45 min.
  7. Plate 200 µL onto plate with appropriate antibiotic.


TSS (50 mL)

  • 5g PEG 8000
  • 1.5 mL 1M MgCl2
  • 2.5 mL DMSO
  • LB to 50 mL

Filter sterilize (0.22 µm filter) and store at 4 ˚C or -20 ˚C.

Personal tools