RNA gels - staining and denaturing

Cathy Sprankle Sprankle at ciit.org
Mon Jul 17 21:18:27 EST 1995


In article <avf-1407951358100001 at original-ray.niddk.nih.gov>,
avf at helix.nih.gov (Anthony V. Furano) wrote:

> In article <1995Jul13.233353.11895 at emba.uvm.edu>, amarion at moose.uvm.edu
> (Amy Marion) wrote:
> 
> > Dear netters;
> > 
> >         I need some advice on RNA gels.  I have made 3 attempts to run
> > total RNA through glyoxal/DMSO gels and one attempt with a formamide gel.
> > In all cases I have been unable to see the RNA after ethidium bromide
> > staining.  Even the RNA markers do not show up.
> > 
> > Should RNA be stained with ethidium bromide at all?  (These gels will be
> > used for Northerns.)
> > What is the proper procedure for staining RNA gels (either glyoxal or
> > formamide) with ethidium bromide?
> > 
> >         I have run the RNA through 0.1% SDS, 1.2% agarose gels.  After
> > staining with EtBr all the RNA (including the RNA markers) are visible.
> > Will the 0.1% SDS denature the RNA?  If yes, can the RNA from this gel be 
> > transfered to nylon for a Northern?
> > 
> >         Thanks for your help.
> > 
> > Amy Marion
> > marion at smtplink.ipfw.indiana.edu

I run a lot of formaldehyde gels, and if I am going to stain them, I soak them
for 5 minutes in a 0.5 ug/ml solution of ethidium bromide in water, followed by
a 30 min-2 hr (whatever's convenient) destain in water.  I don't know why your
gels didn't stain--check the concentration of your EtBr stock.  I've never heard
of EtBr going bad, but I guess that's possible, too.  However, I don't
usually stain gels that I'm going to transfer.  I seem to remember
somebody telling me long ago that it interferes with the transfer or the
hybridization or something.  Here is a procedure for methylene blue
staining of the Northern blot (after transfer).  Not only does it tell you
that your RNA is okay, it also tells you that your transfer is okay.

   Staining solution:
   45 mls 3M NaAcetate pH 5.3
   225 mls ddH2O
   "two matchhead" size aliquot of powdered methylene blue
   Combine and agitate till methylene blue is dissolved.  Stain blot(s) 5
min with shaking.  Destain 5 min in H2O.  Staining does not interfere with
hybridization.  It will fade out after 2-3 hybs.

Good luck!
Cathy Sprankle

-- 
Catherine Sprankle
CIIT
e-mail:  sprankle at ciit.org
Opinions expressed (such as they are!) are strictly mine and do
not reflect those of CIIT.



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