Nuclease, S1 Assay

One unit is the amount of enzyme liberating 1µg (0.033 A260) of acid-soluble nucleotides from heat-denatured DNA per minute at 37ÁC and at pH 4.6

Reagents

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.

Procedure

1. To clean glass tubes (two for each point) add 2ml Substrate B for tests. Include 2 tubes with 2ml substrate B for blanks (no enzyme added) and 2 tubes with 2ml Substrate A (native DNA test).
2. Incubate for 5 minutes before adding the enzyme.
3. Add 0.1ml enzyme dilution
4. Incubate at 37°C for 10 minutes.
5. Stop reaction by adding 2ml 15% perchloric acid.
6. Leave on ice for 10 minutes.
7. Centrifuge on a bench-top centrifuge for 15 minutes at 2000 rpm.
8. Withdraw 3ml supernatant and read A260.

 

Calculation

Where 1242 is a factor derived by dividing the reaction volume, 4.1ml, by the A260 of 1μg, which is 0.033, and dividing by the enzyme sample volume used, which is 0.1ml.