Transforming chemically competent cells (Inoue) protocol

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Revision as of 05:01, 23 October 2009 by Vaishnavi Ananth (Talk | contribs)
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Solutions/reagents:

  • TB buffer cells
  • DNA
  • SOC stored at room temperature
  • plate made with appropriate antibiotic

Equipment:

  • Incubator
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Measure out 25 - 200 µl of TB buffer cells into sterile 1.5-ml microcentrifuge tube (1).
    Allow TB buffer cells to thaw on ice.
    Do not use glass tubes which adsorb DNA.
  2. Add DNA to TB buffer cells.
    Mix solution by pipetting up and down several times.
    Keep volume of DNA less than 5% of the cell volume.
  3. Incubate on ice for 30 mins.
    Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate at 42°C for 30 secs.
  5. Incubate on ice for 2 mins.
  6. Add 4 volumes SOC.
    (not critical)
  7. Incubate at 37°C for 1 hr with shaking at 200 rpm.
    Note: Can also save some time here by reducing incubation to ~45 min.
    Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics.
  8. Plate out 100 - 300 µl of DNA onto plate made with appropriate antibiotic.
  9. Incubate at 37°C for 12 hrs(overnight).

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