EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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The Current DNA-Silver Staining Protocol

Juli 13, 2018

We use this staining protocol because it will fix also the smaller DNA-fragments below 150 bp!
Please see what happens if weaker fixing recipes are used: “Silver Trouble
This staining procedure give also best results with denaturing, 7 M Urea containing gel
s!

[Silver-Trouble]

Our Stock Solutions: (order numbers of functioning chemicals see page: General Silver Staining)

    Fixing Concentrate (5x): 15 g Benzene sulfonic acid (= Phenyl sulfonic Acid) + 120 ml ethanol.
    Fill up to 500 ml with dist. water.
    Fixing diluter: 150 ml ethanol. Fill up to 500 ml with dist. water.
    Washing Concentrate (6x): 1.75 g Benzene sulfonic acid. Fill up to 500 ml with dist.water.
    Silvering Concentrate (5x): 5 g AgNO3 + 1.75 g Benzene sulfonic acid.
     Fill up to 500 ml with dist water.
    Developing Concentrate (5x): 62.5 g Na2CO3. Fill up to 500 ml with dist. water.
    Thiosulfate Concentrate: 500 mg Sodiumthiosulfate (* 5 H20). Fill up to 25 ml with dist. water. Renew this solution once per week.

The Final Solutions:

Fixing solution: 40 ml Fixing Concentrate + 160 ml Fixing Diluter
Washing solution: 100 ml Washing Concentrate +500 ml water
Silvering solution: 40 ml Silvering Concentrate + 160 ml dist. water + 260 ul Formaldehyde.
Developing solution: 40 ml Developing Concentrate + 160 ml dist. water + 260 ul Formaldehyde+ 200 ul Thiosulfate Concentrate
 Stopping and preserving: 75 ml acetic acid + 75 ml glycerol + 600 ml dist.water

The Protocol:

Fixing: 40 min
Washing: 3 x 10 min
Silvering: 40 min
Water: 2 min
Developing: 7 min
Stopping: 3 x 10 min

[DNA-Standards]

Our Hardware:

We are using Pharmacia's (Hoefer)
 “Automated Gel Stainer”.
The solutions are changed and shaked automatically!
The fixing solution is heated slightly (to 50° C, see the left beaker).

Etcrot

The Results:

15% Gel run native with a besic buffer

benzgood

15% Gel run denaturated

Etcrot