Competent E.coli (RbCl method)

• Grow 5 ml ON culture, 37°C, 180 rpm with appropriate antibiotics
• Inoculate 100 ml LB with antibiotics with ON culture 1:100
• Shake at 37°C until culture reaches OD₆₀₀ ~0.5 (usually ~3h), put buffers on ice
• Spin cells at 2000 x g, 4°C, 5 min using two Falcon tubes and resuspend each pellet in 10 ml ice cold TFB I
• After a short incubation on ice spin cells down at 2000 x g, $°C, 5 min
• Resuspend each pellet in 2 ml ice cold TFB II and incubate on ice for 30 min.
• Label 80 1,5 ml tubes and get a little bucket with liquid nitrogen
• After 30 min aliquot in 50 μl aliquots and freeze immediately in liquid nitrogen.
• Store cells at -80°C.

100ml culture yields 80 50μl shots and requires 20ml TFB I and 4ml TFB II

TFB I (pH 5,8)

1. 30 mM Potassium acetate
2. 10 mM CaCl₂
3. 100 mM RbCl
4. 50 mM MnCl₂
5. 15 % (w/v) Glycerol

Adjust to pH 5.8 with dilute (0.2%; 1M) acetic acid. Do NOT overshoot. Filter sterilize.

TFB II (pH 6,5)

1. 10 mM MOPS
2. 75 mM CaCl₂
3. 10 mM RbCl
4. 15% (w/v) Glycerol

Adjust pH to 6.5 with KOH. Do NOT overshoot. Filter sterilize. Buffers can be stored at -20°C indefinitely.