Making electro-competent E. coli

LAB PROTOCOL

• Day 1:  Inoculate LB-medium with E. coli strain to make an overnight culture (5ml)

• Day 2: Inoculate fresh LB-medium (30ml) from overnight culture at 1:100 dilution  (300µl)

• Grow at 37°C to an OD600 0.5-0.7 (for 3-4hrs)

• Pellet by centrifuging (4000rpm for 10 minutes or 6500rpm for 5 minutes) and remove supernatant

• Wash cells with ice-cold distilled water (30ml) and pellet again. Repeat wash step 3x.

• Re-suspend cells in 200µl in 40% glycerol

• Aliquot 50µl of cells in PCR tubes and store at -80°C.