Blocking of repeats for cosmid hybridization

Ola Myklebost olam at radium.uio.no
Fri Mar 11 03:24:28 EST 1994


We are having tremendous problems using human cosmids as Southern blot
probes. The problem, of course, is blocking the repeats. We have tried to
prehybridize the filters and probes with sonicated human DNA, and our last,
most extreme protocol was to 

prehybridize the filter with 100 ug/ml sonicated placenta DNA (sigma D3287)
for 8 h

prehybridize 25 ng cosmid probe to 50 ug COT1 DNA (BRL) for 1 h at 65 C in
50 uL

but still no specific bands can be seen over the high background.

The probe blocking alone is sufficient for FISH, so why doesn't it work for
blots? Probably due to the large amount of target and much larger volumes.

Has anyone had success with this?

-- 
Ola Myklebost                   Email  ola.myklebost at labmed.uio.no
Dept of Tumor Biology
Inst for Cancer Research        Tel +47-2293-4299
The Norwegian Radium Hospital   Fax +47-2252-2421
N-0310 OSLO, Norway



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