Annexure III SOLUTIONS AND REAGENTS A. STOCK SOLUTIONS FOR DNA ISOLATION 0.5M Ethylene-diamine tetra acetic acid (EDTA) (ph=8.0) 1M Tris-Cl (ph=8.0) 5M NaCl solution Red cell lysis buffer (10X) White cell lysis buffer (10X) Sodium dodecyl sulphate (SDS) (10%) (ph=7.2) Proteinase K (20 mg/ml) Phenol-chloroform isomyl alcohol (PCA) (25:24:1) mix. Chloroform-isomyl alcohol (CA) (24:1) mix. 3M Sodium acetate (ph=5.2) Ethanol 70% Ethanol Tris-EDTA (TE) buffer (ph=8.0) 1. 0.5M EDTA (ph=8.0) solution (Volume 1000 ml) 186.1 g EDTA (SRL) (Mol. wt.-372.24) was dissolved in 700 ml of double distilled water by stirring continuously on a magnetic stirrer until dissolved completely. Its ph was adjusted to 8.0 using sodium hydroxide (NaOH) pellets (SRL). The final volume of the solution was adjusted to 1000 ml with double distilled water. The solution was filtered, autoclaved and stored at 4 C. 2. 1M Tris-chloride solution (ph=8.0) (Volume 1000 ml) 60.55 g Tris base (SRL) (Mol. wt.-121.1) was dissolved completely in 400 ml of double distilled water. The ph of the solution was adjusted to 8.0 using concentrated hydrochloric acid (HCl) (Qaligens) drop wise with constant stirring. The final volume of the solution was adjusted to 500 ml with double distilled water. The solution was filtered, autoclaved and stored at 4 C. iv
3. 5M NaCl solution (Volume 500 ml) 146.1 g NaCl (SRL) (Mol. wt.-58.44) was dissolved in 300 ml of double distilled water. The final volume of the solution was adjusted to 500 ml with double distilled water. The solution was filtered, autoclaved and stored at room temperature. 4. Red cell lysis buffer (10X) (Volume 1000 ml) Ammonium chloride (NH 4 Cl) 155 mm Potassium hydrogen carbonate 10 mm 0.5M EDTA solution (ph-8.0) 100 mm 82.9 g Ammonium chloride (SRL) (Mol. wt.-53.49) and 10.0 g Potassium hydrogen carbonate (SRL) (Mol. wt.-100.12) were dissolved in 700 ml of double distilled water. 2.0 ml of 0.5M EDTA (ph 8.0) was added to it and the components dissolved by stirring for 5-10 minutes. Final volume of the solution was adjusted to 1000 ml with double distilled water. The solution was filtered, autoclaved and stored at 4 C. 5. White cell lysis buffer (10X) (Volume 500 ml) 0.5 M EDTA solution (ph-8.0) 2 mm 1 M Tris-chloride solution (ph-8.0) 10 mm 5 M Sodium chloride solution (NaCl) 400 mm 50 ml of 1M Tris-chloride solution, 20.0 ml of 0.5M EDTA (ph=8.0) and 400 ml of 5 M NaCl solution (SRL) were mixed thoroughly and the final volume of the solution was adjusted to 500 ml with double distilled water. The solution was autoclaved and stored at 4 C. 6. 10% Sodium dodecyl sulphate (SDS) solution (ph=7.2) (Volume 50 ml) 5.0 g of Sodium dodecyl sulphate (SRL) was added to 40.0 ml of lukewarm double distilled water. The solution was mixed gently avoiding froth formation till the salt got completely dissolved. Its ph was adjusted to 7.2 using dilute HCl and final volume set to 50 ml. The solution was stored at room temperature. 7. Proteinase K (20 mg/ml) (Volume 1000 µl) 20.0 mg Proteinase K was dissolved in 1000 µl of double distilled water. The solution was stored at -20 C. v
8. Phenol chloroform amyl alcohol mixture (25:24:1) (Volume 500 ml) 250 ml of Phenol saturated in 0.01M Tris-Cl (ph=8.0) (USB), 240 ml of chloroform (Qualigens) and 10 ml of isomyl-alcohol (SRL) were mixed in a dark bottle. The mixture was stirred thoroughly for 30 minutes and stored at 4 C. A layer of 100 ml 0.01 M Tris-Cl was added to the mixture to avoid oxidation of phenol. 9. Chloroform isoamyl alcohol mixture CA (24:1) (Volume 500 ml) 480 ml of Chloroform and 20 ml of Isomyl alcohol were mixed thoroughly by constant stirring in a dark bottle for 30 minutes and stored at 4 C. 10. 3M Sodium acetate solution (ph-5.2) (Volume 100 ml) 40.8 g Sodium acetate trihydrate (SRL) was dissolved in 50 ml of double distilled water. The ph of the solution was adjusted to 5.2 with glacial acetic acid. Volume of the solution was adjusted to 100 ml with double distilled water. The solution was filtered, autoclaved and stored at room temperature. 11. Tris-EDTA buffer (TE buffer) (ph-8.0) (Volume 100 ml) 1000 µl of 1M Tris-HCl solution (ph=8.0) and 200 µl of 0.5M EDTA (ph=8.0) were added to 90 ml of double distilled water. The final volume of the solution was adjusted to 100 ml with double distilled water. The solution was stored at room temperature. B. STOCK SOLUTIONS FOR AGAROSE GEL ELECTROPHORESIS Tris-Borate-EDTA buffer (10X) (Volume 1000 ml) Ethidium bromide (10 mg/ml) Gel loading dye for DNA quantification Gel loading dye to check PCR products 1. Preparation of Tris-borate-EDTA buffer (10X) (Volume 1000 ml) Tris Hydroxymethyl amino methane 0.89M Boric acid 0.89M 0.5M EDTA solution (ph=8.0) 20 mm solution 108.0 g Tris-base (Mol. wt.-121.1) (SRL) and 55.0 g of boric acid (Mol.wt.-61.8) (SRL) were added to 700 ml of lukewarm double distilled water. 40 ml of 0.5M EDTA (ph 8.0) was added to the mixture and it was mixed thoroughly. A final volume of 1000 ml vi
was set by adding double distilled water and the solution was filtered, autoclaved and stored at room temperature. 2. Ethidium bromide (10 mg/ml) (Volume 10 ml) 100 mg Ethidium bromide was added to 10 ml of double distilled water in a dark bottle. The solution was stirred for 1 hour for thorough mixing and later stored at 4 ºC. 3. Gel loading dye for DNA quantification (6X) Bromophenol blue 0.25% Xylene cyanol 0.25% Sucrose 40% 250.0 mg Bromophenol blue (Mol. wt.-669.6) (Pharmacia Biotech), 250.0 mg xylene cyanol (Mol. wt.-554.62) (Pharmacia Biotech) and 40.0 g sucrose (Mol. wt.-342.3) (SRL) were added in 100 ml of double distilled water. The mixture was stirred till the reagents got completely dissolved. The solution was stored at 4 ºC. 4. Gel loading dye for PCR products analysis (Volume 100 ml) Bromophenol blue 0.25% Sucrose 40% 250.0 mg Bromophenol blue and 40.0 g sucrose were added in 100 ml of double distilled water. The mixture was stirred till the reagents got completely dissolved. The solution was stored at 4 ºC. C. STOCK SOLUTIONS FOR SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP) ANALYSIS 40% Polyacrylamide gel solution 10% ammonium persulphate (APS) solution N, N, N, N, -tetramethylethylenediamine (TEMED) Formamide dye 1. 40% polyacrylamide gel solution (19:1) (Volume 100 ml) 38 g Acrylamide (Mol.wt.-71.08) (SRL) and 2.0 g of N, N -methylene bis acrylamide (Mol.wt.-154.1) (SRL) were added in 50 ml double distilled water in a flask and constitutents covered with aluminium foil. The mixture was stirred till a clear solution vii
was obtained. Final volume was adjusted to 100 ml with double distilled water. The solution was vacuum filtered and stored in a dark bottle at 4 ºC. 2. 10% Ammonium persulphate solution (APS) (Volume 500 µl) 50.0 mg of Ammonium persulphate (Mol. wt.-228.2) (SRL) was added to 500 µl of double distilled water in a 1.5 ml microcentrifuge tube. The solution was mixed thoroughly with a pipette and kept at 4 ºC. 3. Formamide dye (Volume 10 ml) Bromophenol blue 0.05% Xylene cyanol 0.05% Formamide 90% NaOH 10 mm 5.0 mg Bromophenol blue (Pharmacia Biotech) and 5.0 mg xylene cyanol (Pharmacia Biotech) were mixed in 9.0 ml formamide solution in a 15 ml centrifuge tube. 10 µl of NaOH (SRL) was added and the mixture was mixed gently for few minutes till a clear solution was obtained. The solution was stored at 4 ºC. and solutions for silver staining 10% Nitric acid 0.4% Silver nitrate solution Sodium carbonate Formaldehyde 10% Acetic acid 6% Glycerol 1. Preparation of 10% nitric acid (Volume 100 ml) 10 ml of Nitric acid (Ranbaxy) was dissolved in 90 ml of double distilled water. The solution was stored at room temperature. 2. Preparation of 0.4% Silver nitrate solution (Volume 1000 ml) 4.0 g Silver nitrate (Mol. wt.-169.88) (SRL) was dissolved in 1000 ml of double distilled water and stored at room temperature in a dark bottle. viii
3. Developer (Volume 1000 ml) Sodium carbonate 3% Formaldehyde solution 0.0054% 30 g Sodium carbonate (Mol. wt.-105.99) (SRL) was added to 1000 ml of double distilled water and the solution was mixed thoroughly. 54 µl formaldehyde (Qualigens) was added to the solution just before being used in silver staining. 4. Fixative/stop solution (Volume 200 ml) 10% Acetic acid was used as a fixative or stop solution. For this, 20 ml of glacial acetic acid (Ranbaxy) was added to 180 ml of double distilled water. The solution was stored at room temperature. D. SOLUTIONS FOR DNA SEQUENCING 1. 4% gel solution for DNA sequencing (Volume 150 ml) Reagent Urea 166 mm 10X Tris-borate-EDTA solution 1X 40% Acrylamide solution 4% 54 g Urea (Mol. wt.-60.06) (SRL) was added to 100 ml double distilled water in a flask and covered with aluminium foil. To this solution, 15 ml of 1XTBE and 15 ml of 40% acrylamide solution were added and mixed gently. Final volume of the solution was adjusted to 150 ml and it was mixed thoroughly. The solution was vacuum filtered and stored in a dark bottle at 4 ºC. 2. Loading dye for DNA sequencing reactions (Volume 1.0 ml) Blue dextran 0.05% 0.5M EDTA solution 25 mm EDTA Formamide 80% Blue dextran EDTA buffer was prepared by mixing 250 mg of blue dextran (Pharmacia Biotech) in 5 ml of 25 mm EDTA solution. To prepare loading dye, 800 µl of formamide solution was mixed with 200 µl of blue dextran EDTA buffer and mixed thoroughly. Loading dye was always prepared fresh for the sequencing reaction. ix