Preparing chemically competent cells

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Revision as of 11:03, 27 July 2005 by ClarkeS (talk | contribs) (Use: added detail)
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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 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 4^o} C.
  3. Remove supernatant and replace with 10% original volume TSS.
  4. Make 100 uL aliquots and store at Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle -80^o} C.

Use

  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 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 42^o} C.
    • 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 Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 37^o} C on shaker.
    • Note: Can also save some time here by reducing incubation to ~45 min.
  7. Spread 100-300 µL onto a plate made with appropriate antibiotic.
  8. Grow overnight at 37 °C.
  9. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in less medium and plate it all. This way you will find even small numbers of transformants.

Buffers

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.