about transformation of DNA into E.Coli

Paul N Hengen pnh at ncifcrf.gov
Fri Aug 9 16:46:38 EST 1996


Trevor Bezdek (trevor at leland.stanford.edu) wrote:

> I tried Sailesh's method last night and got a number of colonies, probably
> not as many as standard protocol but I'll have to do a side-by-side check
> tonight since I was also trying new competent cells at the time.  I simply
> added 2 microliters of DNA from my ligation to 50 microliters competent
> cells, left them for 3 minute on ice and then plated half of the cells on
> an LB-amp plate prewarmed at 37° for 15 minutes.  It worked...
> 
> trevor

I just tried the exact method posted on the net, including the 1 hour warming
period for LB plates + 200 ug/ml Carb., but I did not use ligation buffer. I
used 20 ul of frozen comp. DH5aF' cells purchased from BRL (made by the Hanahan
procedure) and 1 ul of 100 ng/ul pUC19 DNA. The heat shock (42C for 2 min)
plate plus SOC expression for 1 hour at 37C had several thousand colonies on it
and the NO heat shock one had about 2-fold less colonies on the plate.

--
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