PROBLEM: DNA Sequence Orientation

z z-suldan at ski.mskcc.nospam.org
Wed Sep 16 07:28:31 EST 1998


In article <35FF3AD8.289BD95C at emory.edu>, "Rick A. Bright"
<rbright at emory.edu> wrote:

> Please Help....
> 
> I have a plasmid vector that contains a region that is supposed to be
> read AS, but I have it reading sense.  Is there a simple way to just
> switch the orientation of this region (40bp)?  I do not have the
> original plasmid to cut from again, so I have to use the one I currently
> have with the wrong orientation.
> 
> Thanks for any suggestions.
> 
> Rick Bright

Off the top of my head, seems your best option is to PCR the fragment and
reclone it -- PCRing is actually not such a bad idea since it gives you
the opportunity to add convenient sites/tags to either end of your
fragment. Otherwise, look at the multicloning site sequence for the vector
that your fragment is cloned into, you might find compatible enzyme site
on either side of the inserts to do a flip   (e.g. Sal1/Xho1  or
BamH1/BglII) or sites that will flip the insert if you go into another
vector. It's just that with such a short frag, you may find it hard to gel
purify and/or kill the enzymes.

Good luck,
Zal



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