Fong:Electrocompetency

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