genomic southern help

han han at biogate.com
Fri Feb 27 14:51:49 EST 1998


Hi,

I got problem in my genomic southern. I used a cDNA probe which PCR
amplified from a cloned plasmid and then gel purified. It's about 100 bp
and located in one exon of my target gene. Probe labeling is performed by
Amarsherm repi random kit. I can get nice signal from my plasmid control
but not my genomic digests. I am wondering if the following steps have
problem: (1)UV cross link of nylone membrane: I use 304 nm UV
transluminator to expose 1 minute. (it works for my PCR product and
plasmid). Is there anyone know the optimal cross-link condition in your
hands? (2)probe is too short? My target gene has 5 short exons
(40bp-120bp). I am wondering if I can use the whole cDNA sequence as
probe? It is said 30-50 nt overlap is enough for a stable hybridization.
However I wish to get some advise from whom have done tons of southerns.

Thanks in advance.

Han





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