CHAPTER 7: REAGENTS AND SOLUTIONS

Transcription

1 7.1. ANALYSIS OF MODULATION OF SOD ENZYME Acetic acid (cat. no , Glaxo Qualigen, India): Bovine Serum Albumin stock solution (BSA, 1mg/ml): 1 mg of standard bovine serum albumin (cat. no. A2153, Sigma, USA) was dissolved in 1 ml of The stock solution was aliquoted and stored at -20 C. 24µl of stock BSA solution was made up to 1 ml with 0.15 M NaCl to obtain working solution at the concentration of 24µg/ml. Butanol (cat. no , Merck, India): Citric acid (0.6%): 0.6 g of trisodium citrate (cat. no. RM1415, Himedia, India) was dissolved, made up to 100 ml with DIDW and stored at 4 C. Dipotassium hydrogen phosphate solution (0.1M): 1.75g of dipotassium hydrogen orthophosphate (cat. no. RM168, Himedia, India) was dissolved and made up to 100 ml with Eosin Y (0.012%): g of Eosin Y (cat. no. E6003, Sigma, USA) was dissolved and made up to 100 ml with deionised distilled water (DIDW) and stored at 4 C. Nicotinamide Adenine Dinucleotide (780µM NADH): 14 mg of NADH (cat. no. RM393, Himedia, India) was dissolved in 25 ml of The solution was prepared freshly each time. Nitroblue tetrazolium (300µM NBT): 26 mg of nitroblue tetrazolium (cat. no. RM578, Himedia, India) was dissolved, made up to 100 ml with DIDW and stored at 4 C. PMS stock solution (1.6mM): g of phenazine methosulphate (cat. no. RM1140, Himedia, India) was dissolved in 1 ml of ethanol and the volume was made up to 25 ml with The solution was stored at 4 C until use. PMS working solution (186µM): 2.9 ml of stock PMS was diluted to 25 ml with The solution was stored at 4 C and brought to room temperature before use. Potassium dihydrogen phosphate solution (0.1M): 1.35 g of potassium dihydrogen orthophosphate (cat.no. RM248, Himedia, India) was dissolved and made up to 100 ml with Potassium phosphate buffer (0.1 M, ph 7.5): 80.2 ml 0.1M dipotassium hydrogen phosphate and 19.8 ml 0.1M potassium dihydrogen phosphate 127

2 solutions were mixed to obtain 100 ml of 0.1 M potassium phosphate buffer (ph 7.5). Sodium chloride (0.15M): g of sodium chloride (cat. no. RM853, Himedia, India) was dissolved, made up to 100 ml with DIDW and stored at 4 C. Sodium cyanide (300 mm): 1.47 g of sodium cyanide (cat. no. S3296, Sigma, USA) was dissolved, made up to 100 ml with 0.1 M potassium phosphate buffer (ph7.5) and stored at 4 C until use. Sodium pyrophosphate buffer (0.052 M, ph 8.3): 2.32 g of tetra sodium pyrophosphate (cat. no. RM6228, Himedia, India) was dissolved in 80 ml The ph was adjusted to 8.3 with 1N HCl, volume was made up to 100 ml with DIDW and stored at 4 C. Standard Superoxide dismutase (4U/µl): 100 µg of standard SOD was dissolved in 100 µl of 0.1M phosphate buffer (ph 7.5). 25µl of stock SOD solution was diluted to 500 µl with 0.1 M phosphate buffer (ph 7.5) to obtain working stock of 0.2U/µl ANALYSIS OF GENETIC VARIATION IN SOD GENE Acrylamide and Bisacrylamide solution (30%): 29 g of acrylamide and 1 g of bisacrylamide was dissolved and made upto 100 ml with Ammonium acetate: 0.78 g ammonium acetate (cat no. MB033, Himedia, India) was dissolved in 300 µl of Ammonium persulphate (10%): 0.1 g of ammonium per sulphate was dissolved in 1 ml of Bromophenol (cat.no.rm117, Himedia, India) Chloroform: (cat. no , Glaxo Qualigen, India) Developer solution: 2.3 g of sodium carbonate and 0.04 g of sodium thiosulphate was dissolved in 100 ml of deionised distilled water and 150 l of 37% formaldehyde was added to the solution. dntp mix (100mM): (cat.no. FC23HL, GeneI, India) Ethanol (80%): 80 ml of absolute alcohol was made upto 100 ml with Ethidium bromide (10mg/ml): 10 mg of ethidium bromide (cat. no. E2028, Sigma, USA) was dissolved in 1 ml of DIDW and stored at 4 C. 128

3 Ethylene Diamine Tetra Acetic acid (0.5 M, ph 8.0): 18.6 g of EDTA (cat. no. E5134, Sigma, USA) was dissolved in 80 ml of DIDW by constant stirring at 50 C. The ph was adjusted to 8.0 with 1N NaOH and the volume was made upto 100 ml with Fixative: 10% ethanol: 10 ml of absolute ethanol was made upto 100 ml with Formamide (cat no. MB012, Himedia, India): Ice cold absolute ethanol: Icecold ethanol (100%): Absolute alcohol was stored at -20 C. Lysis buffer: 100 µl 1M Tris (ph 8.0), 10 µl 0.5M EDTA (ph 8.0), 100 µl 4M NaCl, 100 µl 10% SDS and 10 µl Proteinase K (20mg/ml) were added and made up to 1 ml with M-MuLV Reverse Transcriptase Enzyme: (cat. No , GeneI, India) Nitric acid (0.7%): 0.7 ml of concentrated nitric acid was made up to 100 ml with Oligo dt(18) primer: (cat.no.dt18, GeneI, India) Phenol: Chloroform mix (1:1): 25 ml of tris-saturated phenol was mixed with 25 ml of chloroform. Phenol: chloroform: isoamylalcohol (25:24:1): 25 ml of tris saturated phenol (cat. no. FC3T, GeneI, India) was added to 24 ml of chloroform (cat. no , Glaxo Qualigen, India) and 1 ml of isoamyl alcohol (cat. no , Glaxo Qualigen, India). Proteinase K buffer: 50 µl of 1M tris (ph 8.0), 50 µl of 0.5M EDTA (ph 8.0), 125 µl of 4M NaCl was mixed and the volume was made upto 1 ml with Proteinase K: 20 mg of Proteinase K (cat no. RM MG HIMEDIA, India) was dissolved in 1 ml of RNasin (from human placenta): (cat. no , GeneI, India). Sample loading dye (6X): g bromophenol blue, 0.3ml glycerol and g Xylene cynol FF was added, dissolved and made up to 1 ml with Sodium acetate (0.2 M, ph 4.6): 10 ml of 2M sodium acetate solution was diluted 10 times with DIDW and autoclaved for 15 min at 121 C. 129

4 Sodium acetate (2M, ph4.6): 27.2 g of sodium acetate (cat. no. S9513, Sigma, USA) was dissolved in 80 ml DIDW and the ph was adjusted to 4.6 using acetic acid. The volume was made upto 100 ml with Autoclaved for 15 min at 121 C. Sodium dodecyl sulphate (10% SDS): 10 g of SDS (cat. no. L4390, Sigma, USA) was dissolved in 100 ml DIDW and autoclaved for 15 min at 121 C. SSC buffer (10X): 8.77 g of sodium chloride and 4.4 g of sodium citrate was dissolved in 80 ml of The ph was adjusted to 7.0 by addition of few drops of 10N HCl. The volume was adjusted to 100 ml with The solution was sterilized by autoclaving at 121 C for 15 min. SSC buffer (1X): 10 ml of 10X SSC buffer was diluted to 100 ml in DIDW Staining solution (0.2% silver nitrate solution): 0.2 g of silver nitrate was dissolved in 100 ml of 10 µl of 37% formaldehyde was added. SYBR Green I Master: (cat. no , Roche Diagnostics GmbH, Germany) TAE buffer (10X): 4.84 g of Tris base, 1.15 ml of glacial acetic acid and 2 ml of 0.5M EDTA (ph8.0) was added, dissolved and made up to 100 ml with TAE buffer (1X): 10 ml of 10X TAE was diluted to 100 ml with DIDW Taq DNA Polymerase with buffer TaqA: (cat.no , GeneI, India) TE buffer (ph8.0): 100 µl 1 M Tris-HCl (ph 8.0) and 20 µl of 0.5 M EDTA (ph 8.0) was added and made upto 10 ml with Autoclaved for 15 min at 121 C TEB buffer (0.5X): 10 ml of 10X TEB was diluted to 200 ml with DIDW TEB buffer (10X): 10.4 g of tris-base, 5.5 g of boric acid and 0.92 g of EDTA was dissolved in 80 ml of DIDW, ph was adjusted to 8.3 and the volume was made up to 100 ml with TEB buffer (1X): 10 ml of 10X TEB was diluted to 100 ml with DIDW Tris-HCl (1 M, ph 8.0): 12.1 g of tris base (cat. no. T1503, Sigma, USA) was dissolved in 80 ml of DIDW, ph was adjusted to 8.0 with 1 N HCl and the volume was made upto 100 ml with Xylenecyanol (cat.no.rm859, Himedia, India) 100 bp DNA ladder (Bangalore GeneI, India) 130

5 7.3. SINGLE CELL GEL ELECTROPHORESIS (COMET ASSAY) Alkaline electrophoresis buffer: 120 g NaOH, 3.7 g EDTA and 0.1 g 8- hydroxyquinoline was added and dissolved in The ph was adjusted to 13.5 with 1N NaOH and the volume was made up to 1000 ml with Lysis buffer: 14.6g sodium chloride, 1.2g tris base, 0.37g EDTA, 1g sodium sarcosinate were added, dissolved in DIDW and ph was adjusted to 10 with 1N NaOH. The volume was made up to 89 ml. 10 ml dimethylsulfoxide and 1ml Triton X-100 was added freshly just before use. Neutralization buffer: 4.84 g tris base was dissolved in 80 ml DIDW and the ph was adjusted to 7.5 with 1N HCl. The volume was made up to 100 ml with Phosphate buffered saline: 0.8 g sodium chloride, 0.02 g potassium chloride, 0.14 g disodium hydrogen phosphate and 0.03 g potassium dihydrogen orthophosphate were dissolved in 80 ml The ph was adjusted to 7.4 with 1N HCl. The volume was made up to 100 ml. solution was diluted 10 times. Yo-Pro I (10µg/ml): 10 mg of Yo-Pro was dissolved in 1 ml DIDW and 10µl of stock. 131