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Competent Cell Preparation - (Jan/11/2008 )

I am making competent cells for the first time. I am using a method to make competent cells that calls for adding MgCl2 after autoclaving the media. I forgot to add the Magnesium chloride. I rmemebered only 10 hrs later (overnight) when the media had an OD of 0.022. I went ahead and added the solution at this time. The cells have to grown to an OD of 0.45 Will this affect the preparation of the cells. Should I start over?



there are a large number of difficulties for cloning so i wouldn't add the bad bacteria preparation on them.
Go for another preparation.
ok if you have just to elzectroporate a plasmid to do a mini/maxiprep you can use one tube. Throw axay the rest.


Here's my way to prepare electrocompetent cells:

1. Grow E. coli in 500ml salt-free LB to OD600=0.8.
2. Spin to pellet the cells and discard the medium.
3. Suspend cells in 500ml ice cold diH2O.
4. Spin to pellet cells again and discard the supernatant.
5. Suspend cells in 2.5ml cold 10% sterile glycerol.
6. Aliquot into 100ul and store at -80*C.
7. Check transformation efficiency and should get 10^9 to 10^10 cfu/ug of pUC19.

* Salt-free LB is the trick. It allows one wash to be sufficient. By avoiding repeated washes, it minimizes cell damage and ensures high transformation efficiency. Of course keep all on ice or 4*C.