DNA labeling alternatives

Jarkko Kortesmaa jkortes at paju.oulu.fi
Sat May 27 08:39:08 EST 1995


Klaus Salger (salger at wap4.zi.biologie.uni-muenchen.de) wrote:
: Colin Rasmussen (crasmussen at anat.med.ualberta.ca) wrote:
: : 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.

: : > 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.

I have used PCR-primers (17mer) for labeling. Just omit random primers and
put an excess (e.g. 10-100 ng) of PCR-primer instead. It works, but I
haven't noticed much difference vs. random priming (in cDNA library
screening). 

I agree PCR-primers SHOULD give better (longer) probes, especially with 
short templates. 

Anyway, you CAN label a 600 bp template with random priming, too. 
--
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Jarkko Kortesmaa		Dept. of Biochemistry and Biocenter Oulu
jkortes at paju.oulu.fi		University of Oulu
				Finland					
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