You could try inverse PCR, although this method is not very
useful for just a few plasmid rescues. This would require cutting
genomic DNA with Sau3a or some other frequent endonuclease, ligating the
mix into small circles, and PCR amplifing short sequences flanking the 3'
end of your rosy PZ. Just make primers at the PIR and corresponding
closest site of your chosen restriction site above (i.e. Sau3A) for the
amplification of the ligated rescue.
Perhaps an easier way to rescue the other side of the PZ would be
to partial the DNA with Xba in conjunction with Spe or Nhe1.
Unfortunately this is a very large PZ rescue encompassing KanR ori and
rosy. I have only done this a few times while trying to rescue 5' side.
Brian Ring @ the bio zoo
Bio Unit 1
Florida State University
Tallahassee, Fla. 32306-2043
bring at freenet1.scri.fsu.edubring at bio.fsu.edu
On 24 Aug 1997, Eric C. Liebl wrote:
> Date: 24 Aug 1997 11:05:52 -0700
> From: Eric C. Liebl <liebl at cc.denison.edu>
> To: dros at net.bio.net> Subject: plasmid rescue from both sides of PZ
>> Does anyone know of a restriction enzyme combination that will allow me to
> rescue genomic DNA from the "rosy gene side" of a PZ insert? In
> otherwords, is there a way to rescue genomic DNA on the opposite side of
> that rescued by XbaI cutting?
>> Thanks, ERIC