genomic southern: help needed

han han at biogate.com
Fri Feb 27 17:43:28 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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