Enzymatic Assay of Nuclease S1

1. Objective

To standardize a procedure for the enzymatic assay of Nuclease S1 (N5661).

2. Scope

This procedure applies to the enzymatic assay of Product No. N5661.

3. Definitions

3.1 Purified Water – water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

3.2 Unit Definition – One unit will cause 1.0 microgram of single-stranded nucleic acid to become perchloric acid soluble per minute at pH 4.6 at 37 ºC.

4. Discussion

N/A

5. Responsibilities

Analytical Services laboratory personnel should follow this procedure as written.

6. Safety

Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS: pH = 4.6, T = 37 ºC, A260 nm, Path Length = 1 cm

7.2 METHOD: Spectrophotometric stop rate determination

7.3 REAGENTS:

7.3.1   0.2 M Sodium chloride with 2 mM zinc sulfate and 60 mM acetic acid (Buffer)

7.3.1.1  Prepare 500 mL by dissolving 5.84 g sodium chloride (S9888) 287.5 mg zinc sulfate (Z4750) and 1.85 mL glacial acetic acid (242853) in 450 mL purified water. Adjust the pH of this solution to 4.6 at 37 ºC with 5 N NaOH. Adjust the final volume to 500 mL with purified water.

7.3.2   0.02% bovine serum albumin (BSA)

7.3.2.1   Prepare 100 mL by dissolving 20 mg of bovine serum albumin (A4503) in reagent 7.3.1 (Buffer).

7.3.3   0.06% DNA (Substrate)

7.3.3.1   Prepare by shredding 60 mg of deoxyribonucleic acid type I, sodium salt (D1501) into 50 mL of purified water. Incubate at room temperature for 2 hours. Heat this solution in a boiling water bath with stirring for 20 minutes. Immediately transfer to a PRE-FROZEN 1 L beaker, on ice, and add 50 mL of cold reagent 7.3.1 (Buffer). Prepare fresh and use immediately.

7.3.4   15% Perchloric acid (PCA):  Prepare 50 mL by adding 10.7 mL of perchloric acid (70% solution) (244252) to 39.3 mL purified water.

7.3.5   Nuclease S1 (Enz)

7.3.5.1   Immediately before use, pipette 0.10 mL of Nuclease S1 into 0.90 mL of Reagent 7.3.2 (BSA)

7.3.5.1.1   For Test 1, pipette 0.010 mL of 7.3.5.1 into 2.00 mL of BSA.

7.3.5.1.2   For Test 2, pipette 0.010 mL of 7.3.5.1 into 5.00 mL of BSA.

7.3.5.1.3   For Test 3, pipette 0.010 mL of 7.3.5.1 into 10.00 mL of BSA.

7.4 METHOD

7.4.1   Pipette the following (in milliliters) into suitable glass containers: (perform each test in duplicate for both sample and control)

Place all vials in a suitably thermostatted waterbath and equilibrate to 37 ºC for 5 minutes, then add:

Mix by swirling and incubate at 37 ºC for exactly 10 minutes, then add:

7.4.2   Mix by swirling and place each tube on ice for 10 minutes. Centrifuge all blank and test vials for 15 minutes. Transfer the supernatant from each blank and test level to suitable quartz cuvettes and record the absorbance at 260 nm.

7.5 CALCULATIONS:

 4.1 = Final volume of assay, in milliliters

df = Dilution factor of the specific test

10 = Time of assay, in minutes

 0.10 = Volume of enzyme used in each test

0.033 = A₂₆₀ of 1 μg DNA