Detecting Biotinylated PCR products?

Jim Gerlach GERLACHJ at QUCDN.QueensU.CA
Tue Sep 14 10:48:26 EST 1993


In our efforts to do quantitative PCR we have in the past kinased oligos 
with 32P. Recently we tried using oligos with a 5' biotin group. Following 
separation on a gel and alkaline transfer to a nylon membrane we have been 
visualizing bands with a streptavidin-alkaline phosphatase conjugate and  
chemiluminescence (LumiPhos 530). We have also cut out the bands from the 
nylon membrane and measured the light with a luminometer. All of this worked 
reasonably well and we want to continue using the biotinylated oligos.

What I would like to do is eliminate the transfer step. I have considered 
doing the biotin-avidin step directly on gels (dried or semi-dry) or using 
magnetic beads on the opposing oligo to separate PCR product from primers. 
Any experiences or suggestions would be appreciated. Thanx in advance.
 
Cheers,
Jim
-------
Jim Gerlach                               Cancer Research Laboratories
gerlachj at qucdn (BITNET)                   Queen's University
gerlachj at qucdn.queensu.ca (InterNet)      Room A309, Botterell Hall
tel (613) 545-6446                        Kingston, Ontario
fax (613) 545-6830                        Canada K7L 3N6



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