RNA gels - staining and denaturing

Aaron Pawlyk aaron at ccat.sas.upenn.edu
Tue Jul 18 16:06:29 EST 1995


Here's my advice on the EtBr staining of gels.  Use 1uL of a 500 ug/mL EtBr
solution for each sample to be loaded.  Mix the RNA, loading buffer and EtBr
together.  Spin down.  Heat at 55-65oC for 10-15 minutes.  Chill on ice, spin
down again and put back on ice (spin down in cold room if possible).  Load
gel.  The heating and rapid cooling helps to denature your RNA and keep it
denatured.  It also increases the EtBr staining.  Most of this I discovered
by experimention, help from others and a certain paper:

Effects of Staing RNA with Ehtidium Bromide Before Eletrocphoresis on
Performance of Northern Blots, BioFeedback in BioTechniques, vol. 14, no 6
1993, pp. 932 - 935.

You may also want to check out the following paper.  I don't agree with 
much of what the author says, but he does include many sources:

Review: Southern and Northern Analysis, Richard A. Kroczek, Journal of
Chromatagrophy, 618: 1993, pp. 133-145.



Amy Marion (amarion at moose.uvm.edu) 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




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