Preparation of solutions for DNA extraction

• Chloroform : Isoamyl - Mix 96 ml chloroform and 4 ml isoamyl alcohol and keep alcohol (24:1, v/v) at room temperature in a closed container.
• 10 % (w/v) CTAB - Add 10 g of CTAB to approximately 70 ml of water. Dissolve the detergent by warming the solution to 65 ° C. Adjust the volume to 100 ml.
• 0.5 M EDTA (pH 8.0) - Add 93.05 g of ethylenediaminetetraacetate. 2H₂O to 400 ml of H₂O. Add – approximately 10 g of NaOH pellets to adjust the pH to 8.0 (The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH). Adjust the volume to 500 ml and sterilize by autoclaving.
• 5 M NaCl - Dissolve 146.1 g NaCl in 400 ml water. Adjust the volume to 500 ml and sterilize by autoclaving.
• Phenol : Chloroform : - Mix 750 m l buffered phenol (pH approx. 7.8), 720 m l Isoamyl alcohol (25:24:1) chloroform and 30 m l isoamyl alcohol. Mix by vortexing and keep at 4 ° C in coloured container ( or this could be prepared just before use).
• 1 M Tris - Dissolve 60.55 g Tris base in approximately 300 ml water. Adjust the pH to 8.0 by adding HCl. Adjust the volume to 500 ml and sterilize by autoclaving.
• DNA extraction buffer - 100 mM Tris-Cl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl, 2.0 % (w/v) CTAB, 0.2 % (v/v) 2-Mercaptoethanol