Re Bcl1

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Sep 16 17:12:39 EST 1992


In article <Buow4D.AEI at mentor.cc.purdue.edu>, miguel at aclcb.purdue.edu (PHILLIP_SANMIGUEL) writes:
>In article <920916134440.6044 at UTBC01.CM.UTEXAS.EDU>, READY at UTBC01.CM.UTEXAS.EDU writes:
>+mdv6n at dayhoff.med.Virginia.EDU (Mark D. Van Cleve) writes:
>++Anyone else have trouble with this enzyme?  Either it goes bad very quickly
>++in our freezer, which would be odd for a heat stable enzyme, or we're not
>++using it correctly, or something, because BclI from several different 
>++sources has very little interest in cutting plasmid for us, even when it
>++will consent to cut lambda, which is not always.  Any clues out there?+
>+
>+Mark-
>+	I don't know if this is your problem, but Bcl1 is sensitive to DNA
>+methylation by the (aptly named) _dam_ methylase. Dam+ _colis_ put a
>+N6-methyladenine in the sequence GATC, which is part of the Bcl1 recognition
>+sequence. Won't cut worth a darn in most of the common lab strains. I had this
>+problem several years ago with some DNA containing synthetic linkers I had made.
>+The original construct cut fine, but once it was cloned into _coli_ and
>+reisolated it wouldn't cut at all. Made me feel a little silly, once I figured
>+out what the problem was.
>+
>+Regards,
>+
>+Mike R.
>
>  Yes.  I've heard the reason lambda sites sometimes cut and sometimes 
>don't is that the dam methylase can't always get to the DNA before it is 
>packaged.  
>  Perhaps even more irritating are enzymes blocked by overlapping dam 
>methylation.  XbaI is such an enzyme.  Its recognition site is 
>
>			TCTAGA
>
>If it happens to be followed by a "TC" then the "G" will be methylated.
>Thus there is a 1 in 8 chance any given XbaI site will be methylated  and
>will not cut (1 in 16 for each strand.)  Can be confusing if you're not
>thinking about it. 
>  Do dam-dcm- strains grow poorly?  I wonder why the popular strains are not 
>dam- and dcm-.  
>
>
>                                    _            _
>__________________________  _      (_)  ________(_)________________________
>Phillip SanMiguel        _/  \     /  \  Purdue University Juggling Club
>miguel at aclcb.purdue.edu (_)   \   /    |   *Juggling*Unicycling*COMBAT*
>         _               |      x    _ | 1001 Stewart Center, Box 665
>________(_)______________ \_ _ / \ _(_) _West_Lafayette,_Indiana__47907-1001   

Yes, dam-dcm- strains are a bit sick, and also a bit mutagenic. Not 
a great combination. 

By the way, there are a number of enzymes whose sites are sometimes or
always blocked by dam or dcm methylation. The common ones certainly include
BclI, XbaI, ClaI, TaqI as well as a number of others. The New England
Biolabs catalog has a good listing in the appendix (probably others do
also, but I happen to have NEB in front of me).

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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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