Nuclease, S1 - Assay

Buffer: 0.2M NaCl, 0.002M ZnCl₂, 0.06M CH₃COONa, pH 4.6: dissolve 5.844 gms NaCl (MW 58.44), 136mg ZnCl₂ (MW 136.29) and 1.85ml concentrated glacial acetic acid in 450ml reagent grade water. Adjust pH to 4.6 with 10M NaOH. Bring to a final volume of 500ml with reagent grade water.

Enzyme Diluent: Dissolve 40mg BSA in 200 ml Buffer

Substrate: Shred into small fibers, 60mg calf thymus DNA and dissolve in 50ml reagent grade water by standing at room temperature for at least 18 hours. Additional stirring may be necessary to effect soluiton. Remove 10ml DNA solution to 10ml of buffer. This is native calf thymus DNA solution (Substrate B)

Heat the remaining DNA solution in a large Pyrex test tube with a stir bar in boiling water on a heater/stirrer while stirring for 20 minutes. Immediately pour into PRE-FROZEN 1 liter beaker on ice. Mix equal volumes of the DNA solution and cold buffer. This is heat-denatured calf thymus DNA solution (Substrate A). Use as soon as possible to prevent the blank from elevating.

15% Perchloric Acid: Add 21.5ml concentrated perchloric acid (70%) to 78.5ml deionized water.