how transfer the RNA to Nylon membrane

SerAln SerREMOVETHISAln at netscape.net
Wed Mar 13 10:43:09 EST 2002


There is a good protocol in the The DIG System User's Guide for Filter 
Hibridization manual from Roche (p. 56 in the one we use, which is a little bit 
old), chapter: Hybridization Techniques, RNA Dot Blotting.

You probably could find it on the WWW, but i'm posting a brief summary of the 
procedure:


1. Dilute the RNA sample in RNA dilution buffer (DMPC-treated 
water:20XSSC:Formaldehyde 5:3:2)
2. Heat the sample to unfold the posible secondary structures (it's not on the 
manual, but we still do it, aprox 60ºC for 5').
3. Spot 1ul of the sample onto a dry nylon membrane.
4. Fix the RNA to the membrane by UV crosslinking (that's what we do) or by 
baking in an oven at 120ºC for 30'.

Prehybridize and hybridize as you usually do with your northern blots.

If you are doing a northern blot inestead, follow the protocol that you can 
download from Roche: 
http://www.roche-applied-science.com/prodinfo_fst.htm?/prod_inf/manuals/dig_man/dig_toc.htm

hope this help.

Sergio

sen hu wrote:

> can anyone give me the way how to transfer the RNA to the Nylon membrane.I
> am doing the dot-bolt!and I can not detect the RNA!!thanks a lot!
> 
> 
> <http://www.biowww.net/forum/read.php?f=1&i=5353&t=5353>
> 




More information about the Methods mailing list