DNA labeling alternatives

Colin Rasmussen crasmussen at anat.med.ualberta.ca
Wed May 24 13:14:02 EST 1995


In article <3pucc1$a39 at sunserver.lrz-muenchen.de>,
salger at wap4.zi.biologie.uni-muenchen.de (Klaus Salger) wrote:

> marivonne (marivonne at bio.tamu.edu) wrote:
> : Hello all,
> 
> : I am interested in using a PCR fragment (approx. 600 bp) as a probe in
> : Southern blots. In order to label it (32P), which procedure would be more
> : efficient, nick translation or random-primed labeling? What are the pro's
> : and con's of nick translating a PCR fragment for use as a probe? Pro's and
> : con's of random-primed labeling for same purpose?
> 
> : Any advice will be *greatly* appreciated. Thank you.
> 
> I have allways used random priming with good results. It's simple, fast
> and gives good labeling efficiency.
> But in your case I would use the PCR primers rather than random primers
> for labeling. If you know which one is the coding strand you could even
> use just one of them to end up with a strand specific probe.

Random priming gives the highest specific activity, but if your fragment
is very short (100bp or so) then end-labelling the primer with T4 kinase +
32P-ATP might work better.  In your case, with a 600 bp fragment I would
tend to use random priming.

> I think this should work very good but I've never tried it (didn't have
> the primers) so I couldn't compare random vs. "specific" priming.

Editorial comment to Klaus.

If you have never tried it as you say above, then on what basis are you
recommending that our friend use the PCR primers ???

Colin



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