Detecting Biotinylated PCR products?
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.
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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