Principles of Biochemistry (2+1)

Preparation of gel

 

i. Thoroughly clean and dry the glass plates and spacers then assemble them properly. Hold the construction together with bulldog clip. Clamp in an upright position and then apply petroleum jelly or 2% agar (melted in a boiling water bath) around the edges of the spacers to hold them in place and seal the chamber between the glass plates.

ii. Prepare sufficient volume of separating gel mixture (30 ml) for a chamber of about (10x9x0.1 cm) by mixing the following.  15% gel  10% gel Stock acrylamide solution 10.05 ml 13.3 ml Tris – HCl (pH 8.8) 4.0 ml 8.0 ml Water 5.7 ml 18.1 ml [Degas these in a water pump for 3-5 minutes and then add] Ammonium persulphate solution 5% 0.1 ml 0.2 ml 10% SDS 0.2 ml 0.4 ml TEMED 10.0 ml 20.0 ml.Mix gently and carefully pour the gel solution in the chamber between glass plates. Layer distilled water on top of the gel and let it set for 30-60 minutes.

iii. Prepare stacking gel (1%) by mixing the following solutions: Total volume - 10.00 ml Stock acrylamide solution - 1.35 ml Tris – HCl (pH 6.8) - 1.00 ml Water - 7.50 ml Degas as above and then add. ammonium persulphate solution (5%) - 50.00 ml 10% SDS - 0.1 ml TEMED - 10.0 ml

iv. Remove the water from the top of the gel and wash with a little stacking gel solution. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set (30-40 min.).