Klenow Enzyme
ROCHE/10104531001 - Sequencing grade, from Escherichia coli Iysogenic for NM 964
Synonym: Klenow
Product Type: Chemical
biological source | Escherichia coli |
form | solution |
manufacturer/tradename | Roche |
mol wt | Mr 75 kDa |
packaging | pkg of 250 U (5 x 103 U/ml) |
Quality Level | 100 |
specific activity | >5000 units/mg protein |
storage temp. | −20°C (−15°C to −25°C) |
Application: | Klenow Enzyme has been used in blunt end ligation of recyclin-1 (RCY1) gene into pETDUET-GSTHis6-S Tag vector. |
General description: | 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. |
Other Notes: | For life science research only. Not for use in diagnostic procedures. |
Physical form: | Solution of enzyme, 5U/μl, in storage buffer: 50 mM Potassium phosphate, 1 mM dithioerythritol, glycerol, 50% (v/v); pH 7.0 (4 °C) |
Quality: | 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. |
Unit Definition: | 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: Incubation Buffer 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. Incubation Procedure 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). |
RIDADR | NONH for all modes of transport |
WGK Germany | WGK 1 |
Flash Point(F) | does not flash |
Flash Point(C) | does not flash |
activity | specific activity: >5000 units/mg protein |
Storage Temp. | −20°C (−15°C to −25°C) |
UNSPSC | 41106314 |