Northern blots

thetott lintottl at telus.net
Fri Apr 2 11:32:23 EST 2004


Some suggestions......
#1, don't treath with NaOH - its bad for your RNA and not necessary as 
you should have denatured your RNA before running the gel (You did use 
RNA loading dye with formamide and formaldeyde and heated the samples 
before loading?).

#2 - How much weight is on the gel? Sometimes in an effort to 'push' 
more RNA out of the gel people put heavy weights on the gel. This 
results in a colapsing of the gel before the RNA can move out. If this 
is happening you should see more 18s transfering than 28s.

#3 - Leave the transfer longer - sometimes this helps, remove the wet 
paper towels and put on fresh dry ones.

L. Lintott

u.jacobi at ncmls.kun.nl wrote:
> Hallo,
> 
> 
> 
> I am having problems with Northern blots. After the transfer I have quite little RNA on my membrane and quite a lot still in the gel (RNA quality OK) and the 28S rRNA is only very weak/not transferred. (visualized by Methblue staining) The Northerns give very weak signals after hybridisation even for highly abundant genes.
> 
> Can somebody help me with this problem? Has somebody else the same problems?
> 
> 
> 
> What I am doing: Run a 1% formaldehyde/agarose gel, wash the gel in water, then denature in NaOH and neutralize in Tris (skipping the last 2 steps doesn't make any difference) and finally shake the gel in 20XSSC. Prewetting the membrane in water, and 20XSSC. set up the transfer in 20XSSC (capillary) and transfer overnight, followed by UV-crosslinking
> 
> 
> 
> Thank you very much,
> 
> Rike
> 
> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&begin=0




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