Making Mg++ /Ca++ competent E.Coli BL21 cells

Wednesday, June 02, 2004

Description
This protocol explains how to prepare Magnesium/Calcium competent cells for transformation with DNA. It is described using BL21 cells but other E. Coli cells could be used.

Procedure
1: Innoculate a single colony of BL21 into 10 ml of LB media without any antibiotics and grow ON at 37 degrees with vigorous shaking. (NB grow up single colonies on plates from glycerol stock culture streaked out)

2: Innoculate a fresh batch (100 to 200 ml) of LB media at a dilution of 1:100 and grow for 2-3 hours at 37 degrees until the OD 600 is approximately 0.5.

3: Transfer culture to sterile falcon tubes (50 ml) and place in ice for at least 15 mins. Spin gently at 4000 rpm in a pre-cooled rotor for 5-10 mins.

4: Keep everything as cool as possible, pour SN (media) off and add half original volume of 100mM MgCl2. Resuspend cells gently using 1ml pipette and swirling falcon tube. Leave on ice for 30 mins.

5: Centrifuge as above gently resuspending in 1:20 original volume of 85 mM CaCl/15 % glycerol and store on ice for approximately 60 mins.

6: In cold room dispense 100 ul of cells into sterile pre-chilled eppindorf tubes and freeze on dry ice/ethanol mix or liquid nitrogen. Transfer tubes to -70 degrees.

Recipes
LB media: 5g yeast extract, 10g bactotryptone, 5g NaCl, adjust Ph to 7.4 with NaOH.

Supplies
You will require 50 ml falcon tubes, shaking incubator, 37 degree incubator, centrifuge and 1.5 ml eppindorf tubes.

Tips
Highest competency is achieved if the cells are kept between 0-4 degrees at all times. All apparatus and solutioins must be pre-chilled.

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Making Mg++ /Ca++ competent E.Coli BL21 cells