deborah.hailstones at smtpgwy.agric.nsw.gov.au
Mon Jul 14 19:02:30 EST 1997
Anyone have a tried and true protocol fo incorporating 32-P into
probes via PCR. I've been trying to random-prime label a 100 bp
fragment, and my specific activity has been low. I'm interested in
trying PCR labeling to increase the specific activity of my probes.
I wouldn't recommend trying to random prime a 110 bp probe even by
PCR! Besides the fact that the frag is very short so priming will be
an exceptionally rare event, since only 1 in 4 bases is labelled you
will never get much 32P into the newly synthd DNA anyway.
IMO, the best way to achieve better labelling would be to prime
specifically, with your 3' PCR primer... in my experience this
increases the SA of a probe by at least a factor of 10 (tho prob. more
for such a teeny frag). Another option that i have tried with small
probes in the past is to concatenate it, though this may give problems
with formation of crucifix structures etc....
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