NanoBio: Bacterial Transformation using Electroporation

From OpenWetWare

Revision as of 18:47, 12 June 2009 by Heather M. Jensen (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Simultaneous Transformation using Electroporation

If there are electrocompetent cells available to you in the -80°C, thaw the cells on ice and begin. If you need to make a fresh batch of electrocompetent cells, use either the small-scale or large-scale * Prep for Electrocompetent cells.

    1. Chill cuvettes for at least 5 minutes on ice.
    2. Set electroporator to 2.5kV. (Our electroporator is set to 600Ω)
    3. Add 5pg – 0.5ug DNA to cells. Mix by pipetting. Pippet into a sterile e.p. cuvette.
      • Note: This DNA should be salt-free, so when purifying with kit, use water.
      • Note: Include enough samples for +/- PCR.
      • Note: The DNA should not sit in the cells for more than 1 minute.
    4. Make sure to dry the cuvette to prevent arcing!
    5. Place the DRY cuvette into the sample chamber. Apply the pulse by pushing the pulse button twice. Remove the cuvette.
      • Note: If you hear a popping sound, this means that the sample has arced. This could either be (1) too much salt in your sample or (2) a wet cuvette.
    6. Immediately add 1mL of room-temp SOC or LB and transfer to a culture tube
    7. Incubate 1 hr with shaking at 37°C.
    8. Plate out cells on LB+antibiotic. Grow o/n at 37°C.


Return to Protocols.

Personal tools