Preparing chemically competent cells

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Revision as of 12:28, 9 July 2005 by Josh K. Michener (talk | contribs) (Use)
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Preparation

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

Use

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    1. 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.
  4. Incubate cells for 30 seconds at C.
    1. 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 C.
  7. Plate 200 µL onto plate with appropriate antibiotic.

Buffers

TSS (50 mL)

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