RNA gels - staining and denaturing
Anthony V. Furano
avf at helix.nih.gov
Fri Jul 14 13:58:10 EST 1995
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
Formaldehyde gels are reproducible and easy to run and here is a protocol
that we use, adapted from the given references. The RNAs are stained
before loading and the migration of the RNAs is easy to follow during the
run with a hand held uv light (short wave length). For Northerns, we use
electro-blotting to transfer the RNA to nylon membranes (zeta probe).
Disclaimer - no affiliation with any producer or supplier mentioned.
Also, 0.1% SDS will NOT denature your RNA.
If you have any questions, contact me by e-mail.
------ PROTOCOL -------
References: for gel
Fourney, R. M., Miyakoshi, J., Day III, R. S., Paterson, M. C. (19??)
Focus, vol. 10, pp. 5-7.
for staining procedure
Rosen, K. M. and Villa-Komaroff, L. (19??) vol. 12, pp. 23-24
10X MOPS/EDTA buffer:
0.2 M MOPS 41.8
0.05 M NaAcetate 6.8
0.01 M NaEDTA(diNa,dihydrate) 3.7
adjust to pH 7.0 and autoclave
for 1% agarose gel: (30ml/ Hoeffer mini gel):
0.3 g agarose
3.0 ml 10x MOPS/EDTA buffer
26.0 ml DEP Rx dist. water
dissolve in microwave, cool to 50 C, add 1.53 ml
37% HCHO, pour and let set for 1 hour before use.
NOTE: ADD HCHO AND POUR GEL IN FUME HOOD.
all volumes in microliters
for 1 5 10 20
deionized formamide 10 50 100 200
HCHO 4 20 40 80
10x MOPS/EDTA 2 10 20 40
Ethidium Br (1.7 mg/ml) 0.6 3 6 12
RNA sample* 4 - - -
* This is the volume for 1 20 microliter gel sample;
if the RNA solution
is too dilute, precipitate with NaAcetate and 3
volumes of EtOH, (or better, dry in vacuo) and
dissolve in 4 microliters of H20. One microgram
of markers or sample is more than enough to easily see.
HEAT samples at 65 C for 15 minutes, cool, spin
and load (about 20 microliters / sample slot)
Electrophorese at 80V (milliamps should be 20 - 25.
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