Electroporation

  1. Pre-chill sterile 1.5ml microfuge tubes at –20°C.
  2. Pre-chill .1cm electrode Biorad Gene Pulser Cuvettes at –20°C.
  3. Pre-warm SOC broth to 37°C.
  4. Keep everything on ice until electroporation to prevent damaging the cells.  We use XL1-Blue MRF’ competent cells from Stratagene.
  5. Into the chilled microfuge tubes, add 1ml of ligation and 40ml cells.
  6. It is a good idea to have a negative control (just cells) and a positive control (1 ml of a 1:10 dilution of the pUC 18 DNA control plasmid  [=10pg] that comes with the Stratagene cells).
  7. Put mixture into the bottom of a chilled cuvette.  Tap the cuvette on the countertop a few times to make sure the cells are touching both walls of the cuvette.  That will help prevent arcing.
  8. Shock at 1.7 volts (KV).
  9. Immediately add 960ml of warm SOC broth directly to the cuvette.
  10. Check and record the time constant after each sample to make sure each one received voltage and to see if it stays fairly consistent from sample to sample.
  11. Transfer broth with cells to a sterile 15ml conical tube.
  12. Once all samples have been shocked and transferred to conical tubes, put the tubes at 37°C, 225rpm, for 1hour.  Leave the lids screwed tightly on, and try to angle the tubes in the incubator.  Use tape to hold them in place.
  13. Now they are ready to be transferred back to sterile 1.5ml tubes.
  14. They can be plated immediately or stored at 4°C until later use.
  15.  We usually plate 100 ml and 10 ml of each sample before deciding how much to use when plating out the rest.  Incubate plates overnight.