Analyzing transformants

[Rajesh Singh]

You can choose restriction sites (unique) one within your reading frame
 and one outside reading frame (from your plasmid, again unique i.e., only
 one cutting site). The size of band should give you information about
 the correct orientation of your gene in the clone. Otherwise the
 orientation of the insert is wrong.
 
 best lucks
 
 Rajesh
 
 
 Rajesh Kumar Singh
 European Molecular Biology Laboratory
 C/o DESY, Notkestrasse 85, Geb. 25A
 22603 Hamburg, Germany
 E-mail: rsingh at embl-hamburg.de
 
 On Thu, 4 May 2006, Duncan Clark wrote:
 > Historians believe that in newspost
 > <ZQk6g.22547$vy1.3229 at news-server.bigpond.net.au> on Thu, 4 May 2006,
 > newsnet customer <customer at newsnet.com> penned the following literary
 > masterpiece:
 > >How to check if colonies have my gene in the CORRECT orientation by PCR
 > >and restriction digest? I think the only way is to sequence the region
 > >in both directions.
 >
 > Two PCR's.
 > One vector primer, one 5' insert primer.
 >
 > Same vector primer, one 3' insert primer.
 >
 > Only the PCR with the correct orientation relative to the vector primer
 > will amplify.
 >
 > PCRing up solely the insert using insert primers and an RE digest on
 > that will never give you the orientation.
 >
 > Duncan
 > --
 > I love deadlines. I especially like the whooshing noise they make as
 > they go flying by.
 >
 > Duncan Clark
 > GeneSys Ltd.
 > _______________________________________________
 > Methods mailing list
 > Methods at net.bio.net
 > http://www.bio.net/biomail/listinfo/methods
 
 


		
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