Enzymatic Assay of Nuclease S1, Product No. N5661

1. Objective

To standardize a procedure for the enzymatic assay of Nuclease S1, Sigma Product Number N5661 at Sigma-Aldrich St. Louis.

2. Scope

This procedure applies only to the enzymatic assay of N5661.

3. Definitions

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

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 37ºC.

4. Discussion

N/A

5. Responsibilities

It is the responsibility of trained Analytical Services laboratory personnel to 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 = 37ºC, A260nm, Path Length = 1cm

7.2 METHOD: Spectrophotometric Stop Rate Determination

7.3 REAGENTS:

7.3.1   0.2M 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, Sigma Product Number S9888, 287.5 mg Zinc Sulfate, Sigma Product Number Z4750, and 1.85 ml Glacial Acetic Acid, Aldrich Product Number 242853 in 450 ml purified water. Adjust the pH of this solution to 4.6 at 37ºC with 5N 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, Sigma Product Number 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, Sigma Product Number D1501, into 50 ml of purified water. Let stand 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)

7.3.4.1   Prepare 50 ml by adding 10.7 ml of Perchloric Acid (70% solution,) Aldrich Product Number 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)

  Blank Test 1 Test 2 Test 3
Reagent 7.3.3 (Substrate) 2.00 2.00 2.00 2.00


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

  Blank Test 1 Test 2 Test 3
Reagent 7.3.2 (BSA) 0.10 ------ ------ ------
Reagent 7.3.5.1.1 ------ 0.10 ------ ------
Reagent 7.3.5.1.2 ------ ------ 0.10 ------
Reagent 7.3.5.1.3 ------ ------ ------ 0.10

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

Reagent 7.3.4 (PCA) 2.00 2.00 2.00 2.00

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:

7.5.1. Units/mL enzyme = [A260nm(Sample) - A260nm(Blank)] X 4.1 X df
    (10)(0.10)(0.033)

7.5.1.1   4.1 = Final volume of assay, in milliliters

7.5.1.2   df = Dilution factor of the specific test

7.5.1.3   10 = Time of assay, in minutes

7.5.1.4   0.10 = Volume of enzyme used in each test

7.5.1.5   0.033 = A260 of 1 ug DNA

8. References & Attachments

N/A

9. Approval

Review, approvals and signatures for this document will be generated electronically using DocCompliance (QUMAS). Print a “For Use” copy, if hardcopy with signature verification is required.

Materials

     
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