Gama:Protocol for competent bacteria

Solutions preparation:

Solution A:

• 30 mM KCH3COO

• 50 mM MnCl2

• 10 mM CaCl2

• 100 mM KCl

• 15% glycerol

Solution B

• 10 mM NaMops (pH 7)

• 75 mM CaCl2

• 10 mM KCl

• 15% glycerol

Procedure

Pick single colonies and grow them overnight, at 37°C, in 10 mL of LB liquid media, with agitation (pH 7,5).

Place your 10 mL culture into 400 mL of fresh LB and grow at 37°C above until they reach an optical density of 0,6.

Spin down your cooled culture by centrifuge ate 4000 rpm for 15 minutes.

Then, resuspend the pellet in 100 mL of pre-cooled solution A.

Spin the suspension at 4000 rpm for 8 minutes.

Resuspend the bacteria in 20 mL of pre-cooled solution B.

Freeze the cells in 0,5 mL aliquots and store at -80°C.