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.

Anthony Furano

                    ------    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

Reagents:

10X MOPS/EDTA buffer:
                                               g/l 
                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.

Sample buffer:
                          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.

Electrophoresis:

     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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