Rubidium Chloride method for competent E.Coli

Rubidium Chloride method for competent E.Coli

 

- Inoculate 1ml from an overnight culture into 100 ml Psi broth. Grow at 37 °C with shaking to A550=0.48 (approx. 2,5 – 3 hours).

- Ice 15 min.

- Pellet cells in 4 50 ml tubes at 3500 rpm at 0 °C for 5 mins.

- discard supernatant and resuspend in 0.4 volume TfbI (10 ml per 50 ml tube)

- Ice 15 min.

- Pool 2 tubes and pellet at 3500 rpm at 0 °C 5 min.

- Discard supernatant and resuspend in 0.04 volume TbfII (2 ml per 50 ml tube)

- Ice 15 min.

- Fill out in eppendorf tubes on ice (50 µl/tube) with blue P1000 tip.

- flash freeze in liquid nitrogen or dry ice/ethanol and store at -80°C

 

Psi broth:

 

Per Liter:

Yeast extract:5 g

Tryptone:10 g

Magnesium sulfate:5 g

pH 7.6 with potassium hydroxide

 

TfbI:

 

Per 200 ml:

Potassium Acetate0.588 g(30 mM)

Rubidium Choloride2.42 g(100 mM)

Calcium Chloride0.294 g(10 mM)

Manganese Chloride2.0 g(50 mM)

glycerol30 ml(15 % v/v)

pH 5.8 with dilute acetic acid

 

TfbII:

 

Per 100 ml:

MOPS0.21 g(10 mM)

Calcium Chloride1.1 g(75 mM)

Rubidium Chloride0.121 g(10 mM)

glycerol15 ml(15 % v/v)

pH 6.5 with dilute NaOH

 

Bart-Jan, 30-8-07