about transformation of DNA into E.Coli

ProZyme, Inc. info at prozyme.com
Fri Aug 16 19:28:00 EST 1996


pnh at ncifcrf.gov (Paul N Hengen) wrote:

>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.

>--
>*******************************************************************************
>* Paul N. Hengen, Ph.D.                           /--------------------------/*
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I have long used a similar method for "quick and dirty"
transformations (e.g. reintroducing a plasmid into E. coli from a
mini-lysate).  It works with even carelessly-made CaCl2 comps and the
latter can be stored in the fridge for a week or more.  I simply
incubate a fraction of a microliter of mini-lysate with 20-50
microliters of comp cells on ice for 2-20 minutes and plate.  I don't
prewarm the plates or take any other precautions.

Note, that this works when the selection antibiotic is ampicillin or
chloramphenicol (or, I suspect, with any of the bacteriostatic-type
antibiotics) but not with kanamycin (or, presumably, any of the
bacteriocidal-type antibiotics).  The latter absolutely require
addition of growth medium and incubation to express the resistance
phenotype.

Bruce Amsden




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