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In this article we will discuss about the principle, requirements and procedure for staining of nucleic acid using silver nitrate.
Principle:
Silver salt is reduced to metallic silver which is then deposited in the immediate vicinity of the staining substratum and provides sensitive and reproducible stain.
Requirement:
Dissolve 60 g sodium carbonate in 2 liters double distilled water. Keep in refrigerator. Add 400 µl Na2S2O3 (10 mg/ml) and 3 ml of formaldehyde to prechilled developer just before use.
Procedure:
1. Wash large and small glass plates, kept in 10% NaOH, with double distilled water and wipe with kim wipe papers.
2. Apply 200 µl bind silane to the small plate and spread uniformly with kim wipe paper.
3. Apply and smear 200 µl of repel silane to the large plate and spread evenly with kim wipe paper.
4. Place small plate over the large one with 4 mm side spacers in position and clamp the plates tightly using clips or gel casting clamps.
Casting of gel
5. To a 70 ml of 5% polyacrylamide stock in a conical flask add 400 µl of 10% ammonium persulphate (fresh) and 40 µl of TEMED.
6. Mix well and pour the mixture in the space in between the two plates using a syringe, taking care to see that no air bubbles are caught in between.
7. Place the comb in position and clamp the plates on top and leave it for polymerisation for 12-14 hours.
Electrophoresis
8. Carefully remove the comb, wash wells carefully by gel running buffer using a syringe and needle.
9. With 0.5x TBE pre run the gel for atleast 45-50 minutes at 40 watt.
10. Add equal volume of loading dye to the PCR products. Denature the same at 95°C for 3 minutes and immediately cool on ice.
11. Load samples and run gel at 40 watts until the dye front reach the bottom.
12. Dismantle small plates, remove spacers.
Silver staining:
The steps involve fixing, staining and developing the bands.
13. The plate with gel is soaked in 10% acetic acid for 15 minutes. With mild shaking till the dye disappears and wash the gel in double distilled water for 5 minutes each, twice.
14. Dip the gel in staining solution with shaking for 15 minutes, briefly wash for 10 seconds in double distilled water. The gel must be treated with developer only till the bands appear. Do not allow the gel to become dark.
15. Dip the gel in 10% acetic acid for three minutes to stop reaction and then wash for two minutes twice with double distilled water.
16. Treat the gel with 2% NaOH so that the gel peals out from the sides of the glass plate.
17. Wash the gel in double distilled water, transfer it to Whatman 3 mm filter paper, dry in a drier and preserve.