PCR labelling

Deborah Hailstones deborah.hailstones at smtpgwy.agric.nsw.gov.au
Mon Jul 14 19:02:30 EST 1997


     Brett wrote:
     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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