singh_rajeshk at yahoo.com
Thu May 4 13:10:03 EST 2006
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.
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
> >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.
> 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
How low will we go? Check out Yahoo! Messengers low PC-to-Phone call rates.
More information about the Methods