lacU169

Dr. Duncan Clark duncan at genesys.demon.co.uk
Mon Oct 20 03:47:20 EST 1997


In article <tyr-2-1610971043230001 at news.srv.ualberta.ca>, Karl Fischer
<tyr-2 at bones.biochem.ualberta.ca> writes
>What strain of E.coli do you use to produce the target template for the
>PCR reaction such that it is susceptable to Dpn I digestion (ie. DNA is
>dam methylated)?
>
>GM2163?

You have the wrong way around. All K12 E.coli's are normally dam+dcm+
(B's are always dcm-). There are very few dam- strains around, mostly GM
series. My routine strain for everything is TOP10 (or F') which is
basically DH5alpha plus totally mcrABC- and mrr- and is therefore dam+.
Rather than gel purify the unmethylated PCR product from the original
plasmid template (which will survive PCR to a small degree) we just
found it to be faster and easier to use Dpn I. 

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



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