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
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....
More information about the Methods