MAKING ELECTRO-COMPETENT //E. COLI//

Inoculate 1 liter of 2xYT media with 1/100 volume of an overnight culture of E.coli.

Grow to OD = 0.5-0.7.

Spin at 4,000 g max. for 15 min. in a pre-chilled rotor and pre-chilled bottles.

Resuspend the cells in the original volume of ice cold 1mM Hepes pH 7.0 in water, 5% glycerol.

Spin as before.

Resuspend the cells in half of the original volume of ice cold 1mM Hepes pH 7.0 in water, 5% glycerol.

Wash in 40 ml 10% glycerol, 1mM HEPES pH 7.0.

Resuspend the bacterial pellet in 2 ml 10% glycerol.

Aliquote (50 ml aliquotes) and freeze in liquid nitrogen Store at –80