DNA Polymerase, Taq Assay

Method: One unit incorporates 10 nanomoles of total deoxynucleotides into acid precipitable products with activated calf thymus DNA as primer-template, for 30 min. at 37°C, prepared according to Aposhian and Kornberg 1962.

Reagents
150 mM Tris•HCl, pH 8.5 with 5 mM ammonium sulfate, 24 mM MgCl₂, 30 mM 2-mercaptoethanol and 0.2 mg/ml BSA

Nucleoside Triphosphates: 30 μM dATP, dCTP, dGTP, dTTP spiked with [³H]dTTP to 10⁶ - 2 X 10⁶.

0.5 mg/ml activated calf thymus DNA

10% perchloric acid

1% perchloric acid

Methyl Cellosolve^® (ethylene glycol monomethyl ether)

Enzyme
0.05 - 0.2 units in 5 μl - 10μl Tris•HCl.

Procedure

To clean glass tubes, add 50 µl each of Tris•HCl and DNA. Add 0.1 ml deoxynucleoside triphosphates. Add enzyme 0.05 - 0.2 units in 5 µl - 10 µl volume (for dilution of the enzyme use Tris•HCl). Include one tube without enzyme as a blank. Incubate at 37°C for 30 minutes. Stop reaction by the addition of 1 ml of 10% cold perchloric acid. Filter by suction through cellulose acetate filters, pores 1 µm - 10 µm. Wash four times using 2 ml of 1% cold perchloric acid for each wash. Transfer filtrate to scintillation vials and add 2 ml Cellosolve^® to dissolve filter. Add 10 ml scintillation cocktail and count.

Calculation

units/ml = 3.6 X (reaction cpm - blank cpm)
                    total cpm X reaction volume in ml

units/mg protein =     units/ml        
                                   MgP/ml Lowry