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Silver staining of sequencing gels (sodium carbonate protocol)

 

 

Gel preparation for staining

Dismount the gel running apparatus. Remove one of the two spacers. Gently separate the two glass plates with the aid of a thin metal spatula or a sharp knife (careful: the edge of a glass plate is very brittle). At start, check carefully which plate the gel got attached on. If it’s the upper plate, stop applying pressure, turn the plates upside-down and continue the procedure (if the plates are separated while the gel is attached to the upper plate, it might detach and get damaged).

The 0,5mm sequencing gel will be stained while still attached to one of the glass plates.

 

Staining protocol

Throughout the procedure, there is no need to constantly shake the gel. If it’s properly covered with the solutions, only one or two brief, gentle shakings will be necessary for each step. Harsh agitation will remove the gel from the glass plate and should be avoided. Some care should also be taken while removing the plate from one solution to pour it into the next one, although sitting the plate almost vertically is useful when feasible, to drip off solutions from the glass and gel.

 

This protocol is adapted from a Wendell Lab Oakland University protocol, which was hosted at this location (but apparently isn't there anymore):
 
http://personalwebs.oakland.edu/~wendell/DNASilverStain.pdf
 
You could try googling for "Silver Staining of Polyacrylamide Minigels for Detection of DNA", you might still find it somewhere else.
 
 
The Wendell Lab protocol was in turn a modification of Neilan et al, Biotechniques 17 (4): 708-712 (1994), which unfortunately I can't find online.
 
For personal safety, all steps should be performed under a fume hood.

Nitric acid and glacial acetic acid are corrosive and toxic by contact, ingestion and inhalation.

Silver nitrate is toxic by contact and ingestion, and stains both clothes and skin.

Formaldehydeis toxic by contact, ingestion and inhalation.

 

1)10-minute incubation in fixing solution: 10% Ethanol in dH2O

(100 ml absolute ethanol + 900 ml distilled water)

***start preparing the nitric acid solution as the incubation starts

 

2) 3-minute incubation in nitric acid 0.65% in H2O

(10 ml nitric acid 65% in 990 ml dH2O)

 

3) three washes with distilled water, 5 minutes each

 

4) 15-minute incubation with 0.2% silver nitrate in dH2O

(2 grams of AgNO3 per liter of water)

Can be reused several times; store in dark bottles and protected from light

***start preparing the developing solution as the incubation starts

 

5) one wash with distilled water, 2 minutes

 

6) 5-20 minutes incubation in developing solution (until color appears)

3% sodium carbonate (Na2CO3) = 30 grams/liter

0.02% formaldehyde = 500 ul formaldehyde 37% per liter

Some care should be taken in dissolving sodium carbonate; it can form clumps which are difficult to dissolve. To avoid clumping, it is useful to use only fine-powdered NaCO3, to pour some of the water in the container before the Na2CO3 is added, add the rest of the water and mix to dissolve as soon as possible.

 

7) 5-minute incubation in stop solution (3% w/v acetic acid in dH2O)

30 ml glacial acetic acid in 970 ml dH2O.