>>>Help about formal/agarose gel <<<

S L Smithson bspsls at bath.ac.uk
Thu Oct 20 04:16:55 EST 1994



I have been running a lot of formaldehyde/agarose gels recently (one of these
days a northern blot will work) and I have tried all three recommended methods
of staining RNA.  One method suggested the incorporation of EtBr into the gel,
much as in standard DNA agarose gels.  I found that this did not give me clear
banding in my marker lane.  Another is post-staining of the gel in a solution
containing EtBr, again I found that this did not work. The only method that 
has ever worked satisfactoraly is adding a small amount of EtBr to the sample
directly prior to loading.  I prepare my samples as described in Maniatis:
4.5 ul sample (RNA in water upto 20ug)
3.5 ul formaldehyde
10 ul formamide
2 ul MOPS running buffer
incubate at 68 degrees C for 15 mins, chill on ice then add
2 ul of loading buffer (glycerol bromophenol blue, xylene cyanol)
0.5 ul of 5mg/ml EtBr solution (or whatever stock you keep in the lab, I find
that 0.5 ul is more than enough).

Load onto your gel and off you go. I find that visualisation is accurate,
bands are sharp and there is no need to hang around waiting for staining or
destaining.  Of course EtBr stained RNA doesn't blot very well and if I 
have stained my samples I can't get my probe to stick, but thats life. 

Hope someone else can benefit from my nightmare.

Louise



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