Transforming chemically competent cells (Inoue) protocol

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Revision as of 01:17, 20 November 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. Measure out DNA into sterile 1.5-ml microcentrifuge tube (1).
    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).

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 32 mins

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