Andor J Kiss
akiss at life.uiuc.edu
Fri Apr 2 16:33:01 EST 2004
We use a vacuum blotter and always get excellent transfer. It's worth
the money, plus it's transferred in two hours (start to finish).
"thetott" <lintottl at telus.net> wrote in message
news:rAgbc.903$B16.872 at edtnps89...
> 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
> > 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
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