DNA labelling/detection kits ?

Andrew Sharp biajs at zeus.bris.ac.uk
Wed Jun 30 09:55:51 EST 1999

I need to do some Northern blots to detect an RNA virus in pig gut samples
(Ohhh, Im so lucky). I plan to generate probes (~400bp) by pcr and then
label them up with a non-radioactive kit.

Im looking at the Amersham versions; 
AlkPhos direct (how does the direct label bit work then?)
Random prime with Fluorocien-labelled dUTP (ECL or CDP-star detection)
-this relies on an AlkPhos conjugated antibody to detect the probe.

Im also looking at NEBs NEBlot Phototope kit which uses biotinylated
octomers and biotinylated dATP (as well ? not clear) to label the
probe but then uses streptavidin to detect the biotin-labelled probe and
biotinylated AlkPhos to catalyse the chemiluminescence. If the diagrams
are correct, three biotinylated AP molecules should attached to each
probe-bound streptavidin molecule thereby conferring some 3x amplification
- is this so ?

Any comments would be greatly appreciated.
Andrew Sharp
a.sharp at bris.ac.uk
Visiting Fellow
University of Bristol
Dr. Andrew J. Sharp
Visiting Fellow
Dept. Of Clinical Veterinary Science

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