For 1 litre solution

Urea (AG)

453 gms

10X TBE

50 ml

40%(19:1 ratio) Acrylamide : bisacrylamide

125 ml

Distilled water

(make up to 1 litre)

• The small and big glass plates wiped thoroughly with detergent and acetone and allowed it to dry

• Repel silane is applied to the big glass plate and bind silane is applied to the small glass plate uniformly.

• The spacer (0.4 mm) is kept at the edge of the big glass on the repellent applied side and silane applied side of small glass plate is placed over it.

• Take care to align both the glass plates correctly

• Take 60 ml of gel solution, 250ul (10mg/ml) ammonium persulfate and 80 l TEMED and mix briefly

• Pour the gel between the glass plates and allow it to settle for a minimum of 1 hr.

• Fix the gel plates on to the electrophoretic unit and clean the urea with buffer.

• Prick the gel with 62 well combs.

• Pre warm the gel at 40W for 20 min.

• Then clean the residual urea from the wells by taking 50 ml 0.5 X buffer in a syringe.

• Add equal volume of dye solution to the PCR samples and mix thoroughly.

• Denature the PCR products at 94ºC for 3 min just before loading and immediately keep it in ice.

• Load 4-5 µl of PCR products in each well.

• The running conditions are 2 hrs at 40 constant Watts.

• After gel running is completed, the combs are separated and the big glass plate is removed. The gel sticking onto the small glass plate will be used for silver staining

• Gently place the gel in 10% (v/v) glacial acetic acid for 30 min at room temp.

• Rinse the gel in deionized water twice for about 2 min each.

• Immerse the gel in silver staining solution (250 mg silver nitrate and 375 µL formaldehyde in 250 ml water) for 20 min.

• Pour out the silver stain solution and wash the gel quickly with deionized water with in 10 seconds.

• Immerse the gel in an ice-cold developer solution (10oC) [7.5 g sodium carbonate, 375 µL formaldehyde, and 50µl sodium thiosulfate (10mg in 1ml water) in 250 ml water] until optimal image intensity is obtained. Stop the developing process by immersing the gel in 7.5% ice-cold glacial acetic acid.

• Transfer gel onto the what Mann paper.

• Air-dry the gel or dry using gel drier 70oC for 30 minutes.

• Gel as such can be preserve as record.

• Band scoring can be done by the eye and hand. Bands are sized and matched directly on gels, auto radiographic or photographic films, or photocopies on transparency overlays. By presences and absence of band.

• The data set generated following band scoring and analysis of fingerprints needs to be interpreted according to each particular application.