Problem with human genomic Southern Blot

Eva RC eva at imbg.ku.dk
Fri Oct 4 10:01:56 EST 2002


Hi to everyone.

This is my first time joining the bionet.molbio news. I'm working with
Southern Blot at the moment: my samples are human genomic DNA, and I'm using
a BAC that includes my critical region as a positive control. My PCR probe
is around 800 bp long and highly specific, and I label it by random primed
labelling with 32P-dCTP. I've tried many different hybridization conditions,
including several temperatures and buffer compositions; different digestion
times and several restriction enzymes; different ways of treating the gel
before tranfer; ... and I always get the same result: no band at all for my
samples and exactly the expected band in the lane with the digested BAC.

Would any of you have an idea of what might be happening, or have any
suggestion?
Than you very much.

Best wishes.
EvaRC.






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