Electrocompetent cells preparation

• Overnight: Grow 1 ml culture overnight at 37°C.
• Subculture 1:100 in 100ml 2xYT (or LB) in 125ml flask. Shake at 37°C until 0.4 < OD600 < 0.8 (usually 2.5 hours)
• Pour into two 50 ml falcon tubes.
• Pellet for 5 min at 4000 xg, 4°C.
• Resuspend cells in 50 ml ice cold H2O by gently using 5ml pipette. (Combine into one falcon tube).
• Pellet for 5 min at 4000 xg, 4°C.
• Resuspend cells in 50 ml ice cold 20% glycerol.
• Pellet for 5 min at 4000 xg, 4°C.
• Resuspend cells in enough ice cold 20% glycerol to reach OD600 = 100. (requires measuring OD600 of a 1:1000 dilution in PBS.)
• Aliquot 100 µl in prechilled tubes. Can freeze at -80°C.