genomic sourthern, help needed

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Mon Mar 2 11:28:19 EST 1998


At 19:44 2/27/98 -0800, you wrote:
>>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. 

Yes you can use the entire cDNA as a probe as long as you know the
restriction sites within your cDNA and from your genomic mapping studies,
you also know (as best you can) the restriction sites within you genomic
clones as well.  

Hybond N+ or any charged nylon is likely not the problem.  It is likely
more of an issue of specific activity.

>>However I wish to get some advise from whom have done tons of 
>southerns.
>>



More information about the Methods mailing list