Biotechnology and Bioinformatics (1+1)

Stock solutions

1. Acrylamide (30%): Dissolve 30g acrylamide and 0.8 g bisacrylamide in 70 ml of distilled water and make up to 100 ml with the same.

2. Separating buffer: Dissolve 36.34g Tris in 150 ml distilled water and adjust the pH to 8.8 with con. HCl. Add 0.8g SDS and make up the volume to 200ml.

3. Stacking buffer: Dissolve 12.1g Tris in 150ml of distilled water and adjust the pH to 6.8 with con. HCl. Add 0.8g SDS and make up the volume to 200 ml with distilled water.

4. Running buffer: Dissolve 28.8 g glycerine, 6.1g tris and 1g SDS in 800 ml distilled water and make up the volume to 1 litre.

5. Sample buffer: Mix 3 ml of glycerol, 1.2 ml of 1M Tris (pH 6.9), 200 μl mercaptoethanol and 600μl of 0.02% bromophenol blue and dissolve 1g of SDS in the mixture. The buffer should be prepared fresh and stored at 4°C.

6. Ammonium persulphate (APS): Dissolve 50 mg of APS in 0.5 ml distilled water. This solution should be prepared fresh and stored at 20°C in aliquots.

Gel recipes (Separating gel)- TABLE 2.

Chemicals	7%	10%
Acrylamide (30% Stock) Double distilled water Separating gel buffer TEMED Ammonium persulphate	7ml 15.4 ml 7.5 ml 30 μl 70 μl	10ml 2.4 ml 7.5 ml  30 μl 70 μl

CAUTION

Acrylamide and bis-acrylamide are neurotoxins and should be handled with care