crude bacterial DNA prep

Duncan Clark blackhole at abuse.plus.com
Tue Aug 9 03:35:51 EST 2005


Historians believe that in newspost 
<mailman.302.1123547527.29381.methods at net.bio.net> on Mon, 8 Aug 2005, 
Michael Sullivan <mlsulliv at wisc.edu> penned the following literary 
masterpiece:
>I want to use PCR to confirm the presence of a large, low copy number 
>plasmid from a bacterial strain.
>
>Can one simply use a small amount of culture directly in a PCR 
>reaction, or should one do some sort of DNA prep, even a crude one, 
>prior to PCR? If the latter, does anybody have any suggestions for an 
>easy prep to make PCR-quality DNA.

For 15 years we have toothpicked 1mm colonies into 20ul water, heated at 
100C for 5mins and used 1ul in the subsequent 50ul PCR.

25 cycles is enough for pUC derivatives and 30 for pET vectors. For 
lower copies try 35 cycles.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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