IUBio

DIG High Prime

John F Mackay etc MackayJF at bmnz.co.nz
Tue Mar 2 19:37:31 EST 1999


Hi Josh,

Make sure you DNA is phenol/chloroform purified...no other way!  Variability
can be due to variable gel extractions etc. whereas phenol/chloroform will
clean it up.
Starting with 300ng clean template will yield 2ug of labelled DNA after
overnight labelling.....= 2ug/22ul   = 90ng/ul. A small aliquot (eg 1-2ul)
is diluted to 100pg/ul and then 1/10 serial dilutions performed. One of each
of these dilutions is spotted out on membrane under a corresponding spot
from the control DNA  (5ng/ul starting conc.)

The other option is as Raymond suggested which is to use PCR labelling.
Simply perform a standard PCR and a PCR with a substituted nucleotide mix
which is:

	products under 1kb    66uM DIG-dUTP (alkali-labile), 133uM dTTP, 200uM
dCTP, 200uM dATP, 					    200uM dGTP

	products over 1kb     33uM DIG-dUTP, 133uM dTTP, others as above

Run aliquots of the labelled vs unlabelled, look for the weight-shift of the
labelled and if the bands are equivalent intensity, simply use 2ul
denatured-and-quenched PCR product directly; per ml of hybe buffer  (DIG
Easy Hyb recommended).

ie, 10ml hybe buffer...take 20ul labelled PCR reaction (make up to 50ul for
easier pipetting), denature, quench and add to hybe buffer.

Check out the paper co-authored by a Roche DIG guru Barbara Rueger which is
about DIG PCR labelling:

BioTechniques   Vol 21 (1996)  1067-1072


Cheers,

John




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