Klenow enzyme is the large, C-terminal fragment (Mr 76,000) of E.coli DNA polymerase I, which can be obtained by subtilisin treatment of intact DNA polymerase I. It retains the 5′→3′ polymerase and the 3′→5′ exonuclease activities of intact DNA polymerase I, but lacks the 5′→3′ exonuclease activity of the native enzyme. The enzyme catalyzes the addition of mononucleotides from deoxynucleoside-5′-triphosphates to the 3′-hydroxyl terminus of a primer/template DNA. This property is used to synthesize DNA complementary to single-stranded DNA templates.
Klenow Enzyme is formed by subtilisin treatment of DNA polymerase I.
Klenow Enzyme has been used in blunt end ligation of recyclin-1 (RCY1) gene into pETDUET-GSTHis6-S Tag vector.
Function test: Sequencing Grade Klenow Enzyme is tested in sequencing reactions according to Sanger. The test is conducted with 1 unit of enzyme and the M13 sequencing system. Sequencing gels are examined by autoradiography. The readable sequence must be ≥250 bases.
Absence of contaminants: Sequencing Grade Klenow Enzyme is tested on various DNA substrates to ensure the absence of nonspecific endonucleases and exonucleases.
One unit is the enzyme activity which incorporates 10 nmol of total nucleotides into an acid-precipitable fraction in 30 minutes under the following assay conditions:
130 mM Potassium phosphate, 6.5 mM MgCI2, 33 μM 14C-dTTP, poly[d(A-T)], 0.833 A260/ml; dATP, 33 μM; 1 mM dithioerythritol; bovine serum albumin, 0.032 mg/ml; pH 7.4.
0.05 to 0.25 units of enzyme are incubated for 30 minutes at +37 °C in a total volume of 300 μl incubation buffer.
Volume Activity: 1,000 units/ml, or 5,000 units/ml respectively, according to Richardson (+37 °C, poly [d(A-T)] as primer).
Solution of enzyme, 5U/μl, in storage buffer:
50 mM Potassium phosphate, 1 mM dithioerythritol, glycerol, 50% (v/v); pH 7.0 (4 °C)
For life science research only. Not for use in diagnostic procedures.