This protocol will create 8 60 ul aliquots of electrocompetent cells per 25 ml of outgrowth culture. I would recommend an alternative protocol if you would like to make competent cells on a large scale.

Adapted from: http://openwetware.org/wiki/Sauer:Electrocompetent_cells

Day 0

Start an overnight culture of the cells that you would like to make electrocompetent

Day 1

  1. Make a 1/100X dilution of your overnight culture into fresh medium (and add antibiotics if desired).
    1. ex. innoculate 24.75 ml of LB with 250 ul of overnight culture
  2. Grow your culture to an OD600 nm ~ 0.5-0.7.
  3. Meanwhile:
    1. Chill sterile water and a sterile 10% glycerol solution at 4°C.
      1. You need 6-7 ml of glycerol per 25 ml of culture-- ex. if you have 2 25 ml cultures shaking, you need 14 ml of 10% glycerol
      2. You need 25 ml of water per 25 ml of culture- ex. if you have 2 25 ml cultures shaking, you need 50 ml of water
    2. Put 1 50 ml conical falcon tube on ice for every 25 ml of culture
      1. ex. if you have 2 25 ml cultures shaking, put 2 falcon tubes on ice
    3. Place 8 "mini-yellow" tubes per 25 ml of culture in a rack in the -80
      1. ex. if you have 2 25 ml cultures shaking, put 16 "mini-yellow" tubes in the -80
  4. Once the correct OD is reached, transfer to the chilled 50 ml falcon tube and leave in ice for 15 minutes
    1. MEANWHILE
      1. MAKE SURE THAT THE CENTRIFUGE IS AT 4 DEGREES. USE THE "FASTCOOL" PROGRAM
      2. Move the water and 10% glycerol to an ice bucket
  5. Centrifuge the cultures at 4000 rpm, 15 min, 4 degrees
  6. Dump the supernatant
    1. MAKE SURE TO KEEP THE TUBES ON ICE WHEN NOT RESUSPENDING THE PELLET OR DUMPING SUPERNATANT
  7. Resuspend the pellet gently with 5 ml of water
  8. Centrifuge the cultures at 4000 rpm, 15 min, 4 degrees
  9. Gently dump the supernatant- it becomes increasingly less solid as you increase spins due to lower salt concentrations 
  10. Repeat steps 7-9 three more times for a total of 4 resuspensions with 5 ml of water+spin+supernatant dump
  11. Resuspend the pellet with 5 ml of 10% glycerol
  12. Centrifuge the cultures at 4000 rpm, 15 min, 4 degrees
  13. Dump the supernatant VERY GENTLY. THE PELLET WILL BECOME LESS SOLID AS TIME GOES ON>> TRY TO DUMP THE SUPERNATANT FROM THE GLYCEROL SPIN ASAP
    1. Note- if you lose a lot of the pellet, do not dispair. Keep going anyways.
  14. Resuspend the pellet in 500 ul of 10% glycerol
  15. Transfer 60 ul to each chilled "mini-yellow" tube
  16. Place in -80
  17. DONE!
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