32-P labeling of PCR products.
zyu at UNIXG.UBC.CA
Wed Jun 11 00:50:55 EST 1997
I would like to label the PCR products during PCR amplification
to minimize any error which can be introduced after PCR reaction.
Random labeling is not my choice for my quantitation of my PCR
products by Phosphoimager.
On Fri, 6 Jun 1997, Andreas Matern wrote:
> In article <Pine.GSO.3.95q.970605133949.15400B-100000 at netinfo2.ubc.ca>,
> zyu at UNIXG.UBC.CA (Zhongtang Yu) wrote:
> > I am trying to throw 32-P dCTP into my PCR tubes to label
> > the PCR product for quantitation by phosphoImager after
> > electrophoresis. Does any one know how much dCTP I should use?
> > A detailed reipe will be more helpful. Any info will be very
> > much appreciated.
> > Zhongtang Yu
> > UBC, Microbiol.
> There are several methods that can be used to radioactively label DNA. We
> use the "random hexamer" method (Feinberg and Vogelstein. 1984. Anal.
> Biochem. 132:6-13)
> 1) Add 70ng probe + H2O to a total volume of 10 ul (generally 5 ul of each)
> 2) Heat in 100 deg C heat block for 7 min to denature DNA
> 3) Cool on ice then ad 11 ul LS and 3 units (1ul) Klenow (Large subunit of
> Pol I)
> 4) Add 50 uCi 32P dCTP (generally 5ul)
> 5) Incubate at least one hour at 37 deg C
> Good luck
> Andreas Matern
> alm13 at cornell.edu
> 266 Emerson Hall
> Dept of Plant Breeding and Biometry
> Cornell University
> Ithaca, NY 14853
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