1. Immediately after the electrophoretic run is terminated, slice the stacking gel from the slab, and place the gel in a glass dish with 200 ml (8mm x 7mm mini-gel) of 40% methanol/10% acetic acid for the length of time indicated in the table below. For convenience, the gel can remain in this first fixative indefinitely. Acetic acid is optional in all fixatives if staining nucleic acids.

2. Follow the steps in sequence 1-13 as indicated in the following table. Use of a rotary or rocker platform is recommended. All steps are performed at room temperature. The oxidizer and silver reagent are supplied as 10x concentrates, and are diluted 1:10 in dH2O just before use. Store the concentrates at 4°C.

                                                  Duration                  
                                      <0.5 mm      0.5-1.0 mm   >1.0 mm     
     Reagent            Volume a         gel          gel          gel
1.  Fixative I          400 ml         30 min        30 min      60 min      
2.  Fixative II         400 ml         15 min        15 min      30 min      
3.  Fixative II         400 ml         15 min        15 min      30 min      
4.  Oxidizer            200 ml          3 min         5 min      10 min      
5.  dH2O                400 ml           --           5 min      10 min      
6.  dH2O                400 ml           --           5 min      10 min                                                                        
7.  dH2O                400 ml           --            --        10 min      
8.  Silver reagent      200 ml         15 min        20 min      30 min      
9.  dH2O                400 ml           --           1 min       2 min     
10. Developer           200 ml         30 sec.  Develop until solution      
                                       turns yellow or until a brown        
                                       "smoky" precipitate appears.  Then   
                                       pour off developer, and add fresh    
                                       developer.                           
11. Developer           200 ml          5 min         5 min        5 min     
12. Developer           200 ml            --          5 min        5 min     
13. Stop solution       400 ml          5 min         5 min        5 min     
                                                            
 -- Indicates that the step is omitted.                            
a Recommended volumes for 16 cm slab gel in a 21 x 21 x 5 cm      
baking dish.  For smaller gels in a smaller container, decrease     
volumes proportionately.  For 8 mm x 7 mm mini-gels, use one-half                                     
the volume shown.

Fixative I (500 ml)                Fixative II (500 ml)

methanol       200.0 ml (40%)      ethanol       50.0 ml (10%)
acetic acid     50.0 ml (10%)      acetic acid   25.0 ml (5%)
dH2O           250.0 ml            dH2O         425.0 ml

Stop Solution (500 ml)             Kit Reagents

acetic acid     25.0 ml (5%)	   1. Oxidizer: Dilute 1:10 with dH2O.
dH2O           475.0 ml		   2. Silver reagent: Dilute 1:10
                                      with dH2O.
                                   3. Developer: Dissolve 16 g per
                                      500 ml dH2O.

Caution: All the kit reagents are poisonous. The oxidizer contains
potassium dichromate and nitric acid, the silver reagent contains silver
nitrate, and the developer contains sodium carbonate and paraformaldehyde.
Wear gloves and lab coat when handling, do not mouth pipette.