Calcium chloride Competent cells Preparation and transformat

Tuesday, November 02, 2004

Description
This is one of the standard protocols to prepare competent cells. In this the cells at a particular density are washed with CaCl2 which later helps in the transformation of plasmids into these cells.

Procedure
Procedure

Competent cell preparation
1) Inoculate a single colony into 5 ml LB. Grow ONT at 37 C in a shaker incubator.
2) Add 1% inoculum into 100 ml LB. Wait till OD reaches 0.6 (~3hrs.)
3) Transfer into 50 ml tubes (pre chilled on ice) and keep on ice for 10 min.
4) Spin 4000 rpm, 10 min @ 4 C. Decant the supernatant.
5) Resuspend in 10 ml of 0.1 M CaCl2 and store on ice for 30 min.
6) Spin 4000 rpm, 10 min @ 4 C. Decant supernatant completely.
7) Resuspend in 2 ml of 0.1 M CaCl2 containing 15 % glycerol.
8) Keep as 200 ul aliquots at 70 C after flash freezing in liq N2.
9) Check efficiency by transforming a standard plasmid (eg pUC19).

Transformation

1) Add plasmid DNA to cells (200 ul) and incubate on ice for 30 min.
2) Give a heat shock to these cells by incubating at 42 C for 45 sec.
3) Keep on ice for 2 min.
4) Add 800 ul LB to the tube and shake at 37 C for 1 hr.
5) Plate 100 ul on LB-Amp plate (considering the plasmid has Amp resistance gene).
6) Grow 16-20 hrs at 37C.
7) After this time E. coli colonies harboring the plasmid will be visible on the plate.

Recipes
Require:
Streaked plate of the required E. coli strain
0.1 M CaCl2
0.1 M CaCl2 containing 15% glycerol
Sterile falcons
Luria Broth, LB-Amp plate

Supplies

Tips
The cells should not pelleted at high speeds or else they will form tight pellets, which will be difficult to resuspend.
After the cells reach the desired OD, always keep on ice.

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Calcium chloride Competent cells Preparation and transformat