RNA gels - staining and denaturing
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
> 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
>In all cases I have been unable to see the RNA after ethidium bromide
>staining. Even the RNA markers do not show up.
>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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