APPENDIX-I. Buffer composition. Working buffer

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1 APPENDIX-I 1. Solutions, chemicals and reagents used for DNA extraction 1. Liquid Nitrogen 2. Cetyl Trimethyl Ammonium Bromide (CTAB) Buffer for DNA extraction: Composition of CTAB Buffer: (i) CTAB (10%) 10g CTAB was dissolved in sterile distilled H 2 O and volume was made up to 100 ml with distilled water. (ii) Sodium chloride (NaCl, 4M) 23.37g of NaCl was dissolved in distilled H 2 O and volume was made upto 100 ml. The solution was autoclaved prior to use. (iii) Tris: Cl buffer (ph 8.0, 1M) g of Tris salt was dissolved in distilled H 2 O and volume was made upto 100 ml and ph was adjusted to 8.0 using 1 N HCl. The solution was autoclaved prior to use. (iv) Ethylene Diamine Tetra Acetic acid (EDTA, 0.5 M) g EDTA was dissolved in sterile distilled H 2 O. The ph of the solution was adjusted to 8.0 using IN NaOH. The volume was made upto 100 ml using sterile distilled H 2 O and the solution was autoclaved. (v) 2-Mercaptoethanol (2%) 2% solution provided by manufacturer was used directly. Buffer composition Component Stock sol. Working buffer Vol. of stock taken to prepare 200 ml buffer 1. CTAB 10% 1.5% 40 ml 2. NaCl 4M 1.4 M 70 ml 3. Tris 1M 100mM 20 ml 4. EDTA 0.5 M 20 mm 8 ml 5. Mercaptoethanol 2% 2% 4 ml 6. Distilled H 2 O ml i

2 Total 200 ml 3. Isopropanol 4. Sodium Acetate solution (3M, ph 5.6) g sodium acetate was dissolved in sterile distilled water ph was adjusted to 5.6 with glacial acetic acid and volume made upto 50 ml. The solution was autoclaved and stored till use. 5. Chloroform: Isoamyl alcohol (24:1) mixture 96 ml of chloroform was mixed with 4 ml of isoamyl alcohol. It was stored in amber coloured bottle % Ethanol 70 ml of absolute ethanol was mixed well with 30ml of sterile water and stored in a stoppered bottle till use. II. DNA purification 1. Phenol: Chloroform: Isoamyl alcohol (25:24:1) 100 ml of Tris saturated phenol was added to a mixture of 96 ml chloroform and 4 ml isoamyl alcohol. The mixture was mixed well allowed to settle to form two layers prior to use and stored in amber coloured bottle. 2. RNaseA (20 mg/ml) solution RNaseA : 20 mg Tris-Cl (pii 7.5) : 10mM NaCl : 15mM Sterile water was added to make the volume to 1 ml. The solution was heated at 100 o C for 15 minutes to inactivate any DNase present and then stored in aliquotes at 20 o C. III. Solvent for DNA Tris: EDTA (TE) buffer (10 mm Tris: 1mM EDTA, ph 8.0) 1 ml of Tris (1M) buffer, ph 8.0 and 0.2 ml of 0.5 M EDTA, ph 8.0 was mixed with sterile distilled H 2 O and volume made upto 100 ml. ii

3 V. GEL ELECTROPHORESIS 1. Agarose gel (1.8%) 1.8 g agarose was added to 100 ml with 1 X TAE buffer, the contents were mixed thoroughly and boiled for 2-5 minutes to dissolve the contents. The mixture was cooled down to 40 o C. The molten gel was casted onto a gel tray with a comb to make wells. 2. Ethidium bromide (10 mg ml -1 ) 10 mg of ethidium bromide was dissolved in sterile water and volume made up to 1 ml. The solution was stored in an amber coloured bottle, at 4 o C. 3. Loading dye (10X) solution 1. Bromophenol Blue 0.25% 2. Xylene cyanol FF 0.25% 3. Glycerol 50% 4. Tris: Acetate: EDTA (TAE) buffer 50 X (stock) solution. ph M Tris base, ph M EDTA, ph 8.0 Glacial Acetic Acid VI. PCR reagents 1. Taq DNA Polymerase A stock solution of 3 units l -1 was provided by the manufacturer (Bangalore Genei) was stored at 20 o C X Assay buffer 10 X PCR assay buffer for Taq DNA polymerase containing 15 mm l - 1 magnesium chloride provided by the manufacturer (Bangalore Genei) was used. Storage was at 20 o C. 3. Deoxyribonucleotide Triose phosphate iii

4 datp (10 mm), dgtp (10 mm), dctp (10 mm) and dttp (10 mm) were mixed in equal volumes and stored at 20 o C till use. 4. Magnesium chloride A solution of 15 mm l -1 provided by the manufacturer, stored at 20 o C, was used. 5. Primer The primer was provided by the manufacturer in a lyophilized form. Based on the molecular weight of a given primer, a solution of 100 M was prepared by adding the required amount of sterile water. Storage was at 20 o C. VII. Chemicals and Materials used Type Material Source Molecular weight markers 100bp DNA ladder 1Kb DNA ladder Fermentas Fermentas Enzymes Kits used Taq DNA Polymerase RNase Dneasy DNA Extraction kit PCR product purification kit Fermentas, Bangalore Genei Qiagen Qiagen Bangalore Genei Dyes Chemicals Ethidium Bromide Xylene cyanol Methylene blue Isopropanol, Iso-amyl alcohol, CaCl 2, NaCl, NaOH, Glucose, Methanol, MgCl 2, Chloroform, Glycerol, Acetic acid, MgSO 4, Formaldehyde, - Mercaptoethanol, Tris, Ethanol, Agarose iv Amersco Sigma Sigma Qualigens, Sigma, SRL

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