in situ hybridization

Greg Tobin tobin at ncifcrf.gov
Thu Mar 21 13:24:43 EST 1996


Our lab is getting into in situ hybridization to identify tissues containing
specific DNA or RNA.  This would entail standard in situ hybridization, 
PCR in situ, and RT-PCR in situ.  There are many, many protocols out there
for doing these experiments.  At first, I would like to try in situ hyb with
a radioactive probe and then move to a fluorescent probe.  The PCR-based assays
would be fluorescent.

Can I get some opinions on how to make the best probe?  How long should it be
for in situ hyb?  Would a random primed or PCR-generated probe have higher
sp. activity than the nick-translation probes?  If the target is 2.5 kb, 
would it be better to use a 2.5 kb probe or an 0.5 kb probe (the smaller probe
might penetrate and bind better).

Any advice would be appreciated.





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