Silver staining DNA

Kanematsu Institute kanemats at ee.su.oz.au
Thu Aug 26 22:10:02 EST 1993


In article <25da46$oi0 at triton.unm.edu> dkim at unm.edu writes:


>that silver staining is as sensitive as radioactivity in detection, at least 
>sometimes.  I was referred to _Analytical Biochemistry_  196:80-83 (1991)
>and was told that this method is now licensed to Promega in a kit for non-
>radioactive sequencing called "Silver Sequence".  I will be getting info on 

In our lab we use a simple silver staining technique
	- 10% ethanol wash 5 min
	- 1% nitric acid wash 3 min ( bromophenol blue -> yellow, xylene
	  cyanol -> blue/green)
	- rinse in MilliQ or very pure H2O
	- 0.2% silver nitrate wash 10 to 30 minutes depending on sensitivity
	  you want (but your background also increases).
	- rinse well (but briefly) in pure H2O
	- develop (with lab lights off or in a darkened container) with
		3% w/v anhydrous sodium carbonate
		0.05% v/v formaldehyde
		developer must be made fresh each time with very pure H2O
	  the trick is to rinse the gel and container with an aliquot of the
	  fresh developer until the solution turns yellow-brown, then add the
	  rest of the developer and wash until you get the intensity and
	  contrast you want.
	- 3% acetic acid wash 2 min
	- 10% ethanol wash 5 min

then the gel can be dried down by various means. This does not work on
agarose gels! There is a separate more complicated method for agarose that I
haven't tried. Nor have I tried the Promega kit. But be warned: proteins
stain as well so if you are staining restriction digests or gels of any
reactions containing enzymes/proteins either remove them first or run a blank
with no nucleic acids to see where the proteins migrate.


>be able to get away from radioactivity.   Daniel Kim

If you have no problems with supply of you DNA or with quantities to load on
a PAGE then silver staining is the way to go. Sensitivity will vary with
development time but this also increases background staining of your gel so
result interpretation becomes more subjective.

Good luck

Albert Catalano
Kanematsu Labs
kanemats at morgan.angis.su.oz.au



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