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

NICHOLAS THEODORAKIS ntheo at welchlink.welch.jhu.edu
Thu Oct 20 12:24:15 EST 1994


In article <CxysG7.Cs7 at bath.ac.uk>, S L Smithson <bspsls at bath.ac.uk> wrote:
>
>
>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

This is my method of choice, also (I saw it in a "Focus" article a long 
time ago), but...

I don't seem to have any trouble blotting (in fact the EtBr makes the RNA 
visible on the blot as well) or probing with this method.  Are you using 
nitrocellulose or a charged-modified nylon?  If you are having trouble 
with blotting and are using NC, I would recommend nylon.
		
						Nick


-- 
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
			Nick Theodorakis
			ntheo at welchlink.welch.jhu.edu
			Johns Hopkins Medical School, Baltimore, MD



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