RNA gels - staining and denaturing

Nancy Alexander alexannj at ncaur1.ncaur.gov
Fri Jul 14 09:19:18 EST 1995


In article <1995Jul13.233353.11895 at emba.uvm.edu>, amarion at moose.uvm.edu 
says...
>
>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.
>
>>Amy Marion
>marion at smtplink.ipfw.indiana.edu
>
I have found formaldehyde gels to be easy to run, they give good results, 
and can be transferrred to membranes.  See Sambrook et al p. 7.43 for an 
overall procedure. I generally use 1% agarose, boil to melt in water, 
cool to 65, add 10x MOPS (heated to 65) to give 1x final, add 8.07ml 
formaldehye (resin-treated) to 150ml total.  My sample prep buffer is 
made up in a batch, kept at -20 until needed, and added to my RNA sample. 
Sample prep buffer: 0.72ml formamide, 0.16ml 10xMOPS, 0.26 ml 
formaldehyde, 0.18ml water.  It's best if you dissolve your RNA in this 
buffer, however, I have added from 5-10ul to 5-10ul of RNA and gotten OK 
results.  Heat sample to 65 for 10 min, quick cool, and add about 5ul of 
blue juice (0.1ml of 80% glycerol, 80ul of saturated soln of bromphenol 
blue).  Load on gel, run, stain with EB and you should see bright 
"bands".  Good luck. 




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