genomic sourthern, help needed
GENYAN at kali.com.cn
Fri Feb 27 21:46:09 EST 1998
>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.
I do not think the problem lies in UV-crossing linking. I always get good hybridization results by transfering DNA to Nylon memberane(Hybond N+)without UV and this memberane can also endure seveal stripings. If you want to use UV,just use 150 mJoule treating with your damp sourthern blot.
Have you checked your probe on polyacrylamide gel? Maybe the specific activity of your probe is comaratively low which hybridize with the high abundant PCR products and still give visible signal in your positive control.
Using radom kit to probe 100bp DNA(too short) is not a appropirate method. You can probe using PCR.
Shanghai Institute of Biochemistry
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