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How to prepare my own competent cells? - as above (Aug/06/2007 )

If no competent cells are provided, how do you prepare your own competent cells? (list the detailed experiment procedures) assuming the availability of glycerol stock of the cell


Do a google search and you will find your answer
But since I am here, I will point you towards OpenWetWare. You will find several competent cell preparation protocols.


here are two protocols, also the simple one works biggrin.gif

-Ned Land-

Here's my protocol:

Start overnight culture in 5ml LB shaking at 37 degrees. Expand into 1L LB and grow cells at 37 degrees with shaking to ABS 600 of 0.5 to 1. Place culture in an ice water slurry for 10 mins. Split culture equally by weight into two ice-cold polypropylene tubes. Leave on ice for 15 mins. Spin cells at 2000rpm/4 degrees/5mins. Decant media. Gently resuspend pellet in residual LB on ice. Add 100ml ice-cold filter-sterilized 0.1M CaCl2. Mix gently. Leave on ice for 20 mins or longer. Spin cells same as above. Decant supernatent. Very gently resuspend pellet in 1ml ice-cold 0.1M CaCl2/15% glycerol. Aliquot into pre-frozen eppies and store at -70 degrees.

Note: I have another protocol which is very similar except that it has the incubation in 0.1M CaCl2 for 24-48 hours and claims the efficiency will go up during the first 12-24 hours. This protocol does not include the freezing however and is intended for immediate use of cells. I'm not sure what freezing the cells after prolonged CaCl2 incubation will do.


I wrote most of the ones on Openwetware, and the one which works reliably is the Top10 competent cell one.


I'm freshman here,this is my first talk,thank U for the protocol