is-pcr

Kevin Gilbride gilbride at bu.edu
Tue Nov 25 15:51:14 EST 1997


Anyone sucessfully doing in-situ pcr? I have been having a great deal of
difficulty generating a direct signal from a solution pcr positive tissue
section. I am using a P.E. thermocycler designed for in-situ, have upped
the MgCl2 concentration, a 2/3 dTTP to 1/3 biotin dUTP and use 5-10 U
taq/50 ul reaction with a hot start. I am using using a prediluted
streptavidin from a Dako quick stain kit (10') after a 15' block in 3%
bsa-pbs to detect. 

My pretreat is right out of the P.E guide, eg. dewax, rehydrate,
proteinase K, 0.2% glycine, dehydrate to 100% EtOH and dry.

I have tried cycling 20-40 cycles, eg. 10'@ 95º; (1'@94º,1.5'@55º,2'@72º);
10'@72º; 4ºƒ.

Anything obvious?
Send me a copy of your detailed protocol?
Spam me?


Any input to this situation would be welcome.

KJ Gilbride
Mallory Inst. of Path./ BUSM



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