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