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NUCxyme™ - Assay
Source:
Komagataella phaffii (previously P. pastoris)
CAS:
9025-65-4
EC:
3.1.30.2
NUCxyme™ ASSAY FOR RECOMBINANT SERRATIA MARCESCENS NUCLEASE
Method
A modification of the method developed by Kunitz (1950) based upon the increased absorbance at 260nm observed during the depolymerization of DNA. A standard enzyme preparation should be run in parallel with an unknown because standardization of DNA preparations and their degree of polymerization in solution is not possible.
Unit Definition: One unit causes an increase of 0.007 A260 per minute per ml while acting on highly polymerized calf thymus DNA at 25°C and pH 8.0 under the specified conditions.
One Worthington unit as defined above is equal to 1-2 Serratia marcescens nuclease units as measured by other manufacturers based on the digestion of salmon sperm DNA to acid- soluble oligonucleotides.
Reagents
1.0M Sodium acetate buffer, pH 5.0: Add 5.75ml glacial acetic acid to 80ml reagent grade water. Mix and adjust pH to 5.0 with 5N NaOH. Bring to a final volume of 100ml with reagent grade water.
0.1M Magnesium sulfate: Dissolve 616.2mg Magnesium Sulfate, heptahydrate (FW 246.5) in 20ml reagent grade water and bring to a final volume of 25 ml.
Stock DNA Substrate: Dilute 12.5ml of 0.1M magnesium sulfate to 200ml with reagent grade water. Add 10mg calf thymus DNA (Worthington code: DNA) that has been shredded into small fibers using clean handling and mix by gentle inversion. Let stand overnight at room temperature. If the DNA does not go into solution overnight, stir gently to effect solution. Add 25 ml of 1.0M acetate buffer, pH 5.0 and dilute to a final volume of 250ml with reagent grade water. This substrate solution may be prepared in larger batches and stored for up to 2 months at 0-4°C.
1.0M Tris: Dissolve 12.1g Tris(hydroxymethyl)aminomethane (FW 121.14) in 80ml deionized water. Adjust final volume to 100ml with deionized water.
10mM Tris-HCl, 1mM MgCl2, pH 8.0: Dissolve 1.21g of Tris(hydroxymethyl)aminomethane (FW 121.14) and 0.203g of Magnesium Chloride, hexahydrate (FW 203.3) in 900ml reagent grade water. Adjust pH to 8.0 with 5N HCl. Bring to a final volume of 1000ml with reagent grade water.
Enzyme Diluent: Dissolve bovine serum albumin at a concentration of 1mg/ml in above 10mM Tris-HCl, 1mM MgCl2, pH 8.0 as needed. Unused enzyme diluent may be stored for up to a month.
pH-Adjusted DNA Substrate: On the day of assay, remove an aliquot of stock DNA substrate sufficient for all testing to be done from the cold room and allow to come to room temperature. Adjust pH to 8.0 with 1.0M Tris. Allow pH-adjusted substrate to stand at room temperature for at least an hour before use.
Enzyme
NUCAR is supplied in buffer containing 50% glycerol. Read A280/ml, diluting in reagent grade water as necessary.
mgP/ml = A280/ml x 0.554 x Dilution
Dilute standard and product in enzyme diluent immediately before assay to achieve rates of approximately 0.008 - 0.018 ΔA/min.
Procedure
Adjust spectrophotometer to 260nm and 25°C. Pipette 0.8 ml of pH-Adjusted substrate into cuvettes and incubate in spectrophotometer at 25°C for 3-4 minutes to establish blank rate, if any, and to reach temperature equilibrium. Add 0.2 ml of diluted standard to each cuvette and record A260 for 8-10 minutes. Calculate ΔA260/min from the linear portion of curve which may follow a lag, particularly at higher dilutions.
Using the diluted sample(s) to be tested, repeat the above procedure.
Calculation
Calculate the factor for the standard:
Factor = Activity of Standard / ΔA260/min of Standard x Dilution of Standard)
Determine an average standard value from all acceptable standard dilutions that were tested. Activity of the tested samples are determined using this average standard value:
u/ml = ΔA260/min of Sample x Dilution of Sample x Average Standard Value
NUCxyme™ Products
Description
Activity
Code
Cat. #
Size
Price
Description
NUCxyme™, DNA/RNA Nuclease, Recombinant, AF
Source:
Komagataella phaffii (previously P. pastoris)
Recombinant Serratia marcescens nuclease produced in Komagataella phaffi. Chromatographically purified. Free of animal derived components and proteases. A liquid preparation in 20mM Tris-HCl, 2mM MgCl2, 20mM NaCl, pH 8.0 and 50% glycerol.
Store at -20°C.
Ice Pack required
Code
NUCAR
Product details
LS01522
25 ku
$125.00
LS01524
100 ku
$350.00
LS01525
Bulk
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