why my Northern blot do not work?

Chris H Lindley chris at scotgate2.demon.co.uk
Fri Jun 19 02:02:02 EST 1998


On 18 Jun 1998 11:19:14 -0700, h0w0926 at UNIX.TAMU.EDU wrote:
>I did my Nothern blot, but it did not have any signals. I know the gene
>is highly expressed by previous differential screening of the cDNA
>library. I followed the protocal of the Amersham booklet with the Hybond
>N+ membrane. That is what I did: transfer the RNA use 20x ssc, briefly
>wash with 2xssc, UV crosslink, store in -4C for about one week,
>pre-hybidize for 3 days, hybridize for 16 hours, wash with 2xsspe 0.1%
>SDS two times @ r.t. 10min, 1xsspe @ 65C for 15min, 0.1xsspe @ 65C for
>about 45min. After I did the RNA transfer I checked gel, there was no
>EtBr Stained RNA left on the gel. The Southern blot I did in the same
>box for hybridize and wash  worked beautifully. So I really can not
>figure out what is wrong. If any one can help me, I will be very
>grateful, thanks!

Hi,

I'm no expert, but why did you wash with 2xSSC after the transfer, and
before crosslinking? Is this part of the Amersham protocol? I thought
it's wise to crosslink before any other steps, to avoid removing the 
mRNAs.

Plus you say that there was no EtBr signal coming from the gel after
the transfer. Was there EtBr stained RNA on the membrane at this point?

It sounds like a bad case of degradation!!!

Cheers
Chris

-- 
/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ 
 Chris H. Lindley              				Yorkshire, UK
 chris at scotgate2.demon.co.uk     Ferg on #os/2 and #os2uk on EFnet
 UK OS/2 Users group http://www.denzil71.demon.co.uk/os2/main.html
_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/



More information about the Methods mailing list