Sauer:Electrocompetent cells

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search

Current revision

For a nice background on electroporation, visit Wikipedia's page.

Preparing electrocompetent cells:

  1. Make a 1/100X dilution of your overnight culture into fresh medium (and add antibiotics if desired).
  2. Grow your culture to an OD600 nm ~ 0.5-0.7.
  3. Meanwhile, chill sterile water and a sterile 10% glycerol solution at 4°C.
    1. For a 1 L culture I'd chill 500 mL of water and 100 mL of 10% glycerol, and this is more than enough.:Marylee 23:04, 27 November 2006 (EST)
  4. Place your cultures over ice, ~15'.
  5. Spin at 4000 rpm, 15', and dump the supernatant.
  6. Gently resuspend the pellet in 1 mL of chilled, sterile water. Transfer to a 50 mL Falcon tube and fill it with more water.
  7. Spin at 4000 rpm, 15', and dump the supernatant.
  8. Repeat this resuspension-spin cycle four times, the final time using your 10% glycerol solution.
  9. Resuspend your pellet in 500 μL of 10% glycerol.
  10. Aliquot 60 μL into eppendorf tubes and freeze at -80°C until use.


Electroporation:

  1. Thaw your aliquot of electrocompetent cells on ice and transfer them to an electroporation cuvette.
  2. Add 1 μL of DNA, or whatever else your introducing, to your cells.
  3. Electroporate according to the specifications of your equipment and sample.
  4. Quickly add 1 mL of medium (lacking antibiotics) to the cuvette and then transfer to an eppendorf.
  5. Cure cells by shaking at 37°C (or whatever temperature you would use to plate your cells) for ~1 hour.
  6. Gently pellet cells by spinning at 4000 rpm, 3'.
  7. Resuspend the pellet in roughly 150 uL of supernatant and plate on selective medium.
Personal tools