Silver Staining of Sequencing Gels

Allergan kedziekm at
Fri Aug 5 12:32:08 EST 1994

I had a request to post my protocol for Silver Staining of Sequencing 
Gels.  It is the protocol from Promega that comes with their Silver 
Sequencing System.  The DNA is sequenced by cycle sequencing.  

1.  Before gel is poured, the plates are prepared so that the gel will 
stick to one plate throughout the staining procedure.  It is done with 
BindSilane that comes with the kit.  The other plate I treat with Rain-X.

2.  Reagent quality seems to be important.  I always use MilliQ quality 
water, and ACS grade sodium carbonate.  I use FIsher brand.  It works.

3.  After electrophoresis, the gel is fixed in 10% glacial acetic acid.  
Protocol calls for plastic trays.  Fixing is for 20 minutes with 
agitation (but gel can sit without agitation for longer, even 
overnight).The fixer solution is saved for use later.

4.  Gel is rinsed three times with 2 liters water, for 2 minutes each, 
with agitation.

5.  Gel is stained for 30 minutes, with agitation.  Stain solution is 2g 
silver nitrate and 3 ml 37% formaldehyde in water.  After staining, we 
save the solution and precipitate the silver with 10g sodium chloride.  
We collect the precipitate.

6.  Development.  This seems to be the most critical step. Developer 
solution is 60 g sodium carbonate, 3 ml 37% formaldehyde, 400 ul sodium 
thiosulfate per 2 liters.  I make the carbonate part up the day before, 
and store it in the cold room, adding the formaldehyde and sodium 
thiosulfate immediately before use.  The low temperature of the developer 
seems to help control the rate of development.

	a.  Rinse gel is water before development.  This is called a 
"splash down" - the gel is placed in water only long enough for the water 
to cover it, then removed.  Too long in the water will remove too much 
stain, and bands will not be dark.

	b.  Transfer gel to approximately half of development solution 
and agitate vigorously to prevent background streaking and spotting.  
When upper region of gel, with high MW material becomes visible, transfer 
to fresh development solution.  Continue staining until lower bands are 
visible.  This is a judgement call.  Staining too long will increase 

	c.  Stop development by adding an equal volume of fixer 
solution.  Incubate with shaking for 2-3 minutes.

7.  Rinse stained gel twice, for 2 minutes, with agitation, in water.

8.  Dry (air dry or convection oven).  Dried gel can be read directly on 
gel box, or EDF film can be used to make a permanant record.

This is my procedure.  It works for me - I now do all my sequencing this 
way.  I buy the kit from Promega.  I have no connections to Promega.

Karen Kedzie
Allergan, Inc.

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