Fong:Electrocompetency

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Protocol for the preparation of electrocompetent E. coli stocks

Materials

  • Overnight culture of particular strain (e.g., DH5α, NEB10β, DB3.1)
  • Bucket of ice
  • Refrigerated centrifuge
  • Ice-cold 10% glycerol
  • Chilled, sterile centrifuge bottles
  • Chilled, sterile 15mL conical tubes
  • Chilled, sterile cryogenic vials

Procedure

  1. Inoculate 500mL of fresh LB with a 5mL overnight culture
  2. Shake at 37C and grow until OD600=0.6-1.0
  3. Split cell suspension into two chilled, sterile centrifuge bottles - chill for 20 minutes
  4. Spin at 3000rpm for 20 minutes at ~0C and discard all of the supernatant
  5. Gently resuspend cells in 25mL of ice-cold 10% glycerol
  6. Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant
  7. Gently resuspend cells in 15mL of ice-cold 10% glycerol
  8. Transfer cells to two chilled, sterile 15mL conical tubes
  9. Spin at 3000rpm for 10 minutes at ~0C and discard all of the supernatant
  10. Gently resuspend cells in 2.5mL of ice-cold 10% glycerol
  11. Split cell suspension into 10 chilled, sterile cryogenic vials (250μL aliquots)
  12. Label and store vials in -80C freezer

Notes

  • This stock will be viable for at least 6 months
  • Do not re-freeze aliquots once they have thawed