Erickson:Competent Cell Generation

Generation of Competent Cells

Solutions

• Mg/Ca (3.25 g MgCl₂*6H₂O + 0.6 g CaCl₂*2H₂O), per 200 mL
• 100 mM CaCl₂ (2.95 g CaCl₂*2H₂O) per 200 mL

Procedure

1.Inoculate 100 mL LB with 1 mL of saturated overnight culture (prepare strain needed the day before).
2.Shake at 37°C until OD600=0.4-0.5 (~ 3 hours).
3.Place in ice bath for 5 minutes.
4.Centrifuge at 6000 rpm for 10 minutes in cold SS34 rotor (2 tubes, 40 mL/tube)
5.Decant supernatant.
6.Gently resuspend each pellet with 15 mL of cold Mg/Ca
7.Incubate in ice bath, 30 minutes.
8.Centrifuge (same settings), decant superntant, resuspend each pellet with 1.6 mL of cold 100 mM CaCl2.
9.Incubate on ice for 20 minutes.
10.Combine cells to one tube (total of ~3.2 mL)
11.Add 0.5 mL of 80% glycerol and swirl to mix.
12.Flash freeze (in liquid nitrogen) as 50 or 100 μL aliquots. (Tubes should be labeled beforehand)
13.Store in -80°C in labeled box.

Notes

• All solutions are sterilized by autoclaving.
• Cells should never touch anything that is warm (chill solutions, pipets, tubes, etc.) - DO NOT TOUCH THE TUBES WHERE THE CELLS ARE WITH YOUR HANDS!
• Heat shock conditions are 42°C for 70 seconds.
• Recover transformations in 8 volumes of LB.