Dr. Duncan Clark duncan at
Mon Oct 20 03:47:20 EST 1997

In article <tyr-2-1610971043230001 at>, Karl Fischer
<tyr-2 at> 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)?

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. 

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

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