genomic sourthern, help needed

GENYAN GENYAN at kali.com.cn
Fri Feb 27 22:44:28 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.
>
>Han

Hi,han
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.

Good luck!

-----------------------------------
Genyan Yang
Shanghai Institute of Biochemistry
Academia Sinica
320 Yue-Yang Road
Shanghai, 200031
P.R. China
---------------------------------


___________________________________
Get your own private,FREE email at 
   http://www.kali.com.cn/   
___________________________________




More information about the Methods mailing list