Beta-Agarase and PCR

Vivian Miao vmiao at oregon.uoregon.edu
Sun Mar 7 22:47:05 EST 1993


In article <1993Mar5.212417.23674 at gserv1.dl.ac.uk>, r.nakisa at ic.ac.uk
wrote:

 I cut
> the 1400 ligation product out of a 1% LMP gel and agarase digest.
> Then I spin down the undigested agarose, pipette the supernatent into
> a new eppendorf and precipitate with isopropanol according to the
> instructions.
> 
> But here's the crunch.  When I try to amplify the 1400 b.p. fragment,
> it doesn't amplify at all!  Does anyone know whether Taq polymerase is
> affected by the products of an agarase digestion?
> 

I like the way you thought out your protocol - but since it didn't work,
and
you suspect the agarase, how about just melting the 1.4 kb fragment in its
LMP agarose, and using a tiny bit of that as your PCR template  (say, 1 or
2 5l into a 100 5l PCR reaction)?  This works for me, using Taq.  The two
 main concerns I would have are to dilute the template, and to minimize the
amount of agarose hanging around, just in case.

(Just a thought, in case you're amplifying across the original ligation
site:  
don't bother isolating the ligation product, just use a small amount of the
ligation reaction as PCR template, and let PCR sort out which is the right
ligation product for you!)



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