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Transformation of E. coli: CaCl2-method PCG Solution
 

Summary: Transformation of E. coli: CaCl2-method
PCG Solution
0.76 g Pipes (dipotassium; 378.5 g/mol; 10 mM)
1.76 g CaCl2 (dihydrate; 147.0 g/mol; 60 mM)
30 g Glycerol (at least ACS purity)
Add HCl to pH 7.0. Adjust volume to 200 ml. Autoclave or filter sterilize (50-100 ml aliquots).
NB: LB plus 10 mM MgSO4 and 0.2% glucose can be used instead of SOC medium.
Preparation of competent cells:
1. Dilute a fresh overnight culture 1/100 into 25 ml SOC medium in 250 ml flask. This will yield
enough competent cells for 25 transformations. Grow at 37C until OD600 = 0.5.
2. Place flask with cells on ice for 5 minutes. Transfer to two pre-cooled centrifuge tubes.
3. Centrifuge 20 minutes at 2000 rpm at 4C. (Spin longer if needed to pellet cells.)
4. Gently resuspend pellet in 12.5 ml of ice-cold PGC solution. Hold on ice 30 minutes.
5. Centrifuge 20 minutes at 2000 rpm at 4C.
6. Gently resuspend pellet in 2.5 ml of ice-cold PGC solution. Hold on ice. Competent cells are
stable for 24 hours, and competence usually increases after 12-18 hours on ice.
Transformation of competent cells:
1. Add 100 l of competent cells to a 14 ml round bottom polypropylene tube.
2. Add 0.1 1 g (1-10 l) of transforming DNA. Swirl to mix. Hold on ice for 30 minutes.
3. Heat shock cells for 45 seconds in a 42C water bath. Place tubes on ice for 2 minutes.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine