Vacuum Blotter - RNA transfer

Vellanoweth's Lab jwheele at calstatela.edu
Fri Apr 14 16:15:37 EST 2000


We used 5 min vacuum rinse w/ DEPC water, 5 min vacuum with alkaline solution (50mM
NaOH, NaCl), 5 min vacuum with .1M TrisCl, and then 30 min flooded with SSC on vacuum
blotter.  We used 55 mbar of suction.

Mano wrote:

> By the way, how long did you transfer using the vacuum? I ask because I use vacuum
> blotter.  Thanks
>
> Vellanoweth's Lab wrote:
>
> > We've recently discarded the vacuum blotter for northern transfer.  We are now
> > back to the tried and true papertowel method for blotting which works
> > beautifully and is actually much easier, if a little longer in time.  We found
> > over half our sample was still in the gel with the vacuum blotter and we had
> > bad hybridisation success, now we are hybridising fine, with all the sample
> > there.
> >
> > Janel Wheeler
> > Vellanoweth Laboratory
> > Cal State Univ., Los Angeles
> > jwheele at calstatela.edu
> >
> > Susanne Rohrer wrote:
> >
> > > VASANTHARAJAN JANAKIRAMAN wrote:
> > >
> > > >
> > >
> > > > Even after 2 hrs of transfer at 55mbar, my RNA (Wheat RNA) was still in
> > > > the gel.
> > >
> > > We see the same when using bacterial total RNA . There are lots left in the
> > > gel, but what is transferred is still enough for detection - have you tried
> > > hybridization and detection on it?
> > >
> > > Susanne





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