10104531001

Roche

Klenow Enzyme

Sequencing grade, from Escherichia coli Iysogenic for NM 964

Quality Level

form

solution

specific activity

>5000 units/mg protein

mol wt

Mr 75 kDa

packaging

pkg of 250 U (5 x 103 U/ml)

manufacturer/tradename

Roche

shipped in

dry ice

storage temp.

−20°C (−15°C to −25°C)

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.

Application

Klenow Enzyme has been used in blunt end ligation of recyclin-1 (RCY1) gene into pETDUET-GSTHis6-S Tag vector.

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).

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)

Other Notes

For life science research only. Not for use in diagnostic procedures.

RIDADR

NONH for all modes of transport

WGK Germany

WGK 1

Flash Point(F)

does not flash

Flash Point(C)

does not flash

Ypt31/32 GTPases and their F-Box effector Rcy1 regulate ubiquitination of recycling proteins
Chen SH, et al.
Cellular logistics, 1(1), 21-31 (2011)
Farid El'Garch et al.
Antimicrobial agents and chemotherapy, 51(3), 1016-1021 (2006-12-30)
Screening of a Tn5-Hg insertional library (12,000 clones) constructed in wild-type Pseudomonas aeruginosa strain PAO1 identified four genes (namely, galU, nuoG, mexZ, and rplY) whose disruption individually led to increased resistance to aminoglycosides (means of twofold). Inactivation of these genes...
A Klostermann et al.
The Journal of biological chemistry, 275(50), 39647-39653 (2000-09-20)
Neuronal development and apoptosis critically depend on the transformation of extracellular signals to intracellular actions resulting in cytoskeletal rearrangements. Ena/VASP (enabled/vasodilator-stimulated phosphoprotein) proteins play an important role in actin and filament dynamics, whereas members of the semaphorin protein family are...
Douglas Vernimmen et al.
The Biochemical journal, 370(Pt 1), 323-329 (2002-11-07)
The ERBB2 gene is overexpressed in 30% of human breast cancers and this is correlated with poor prognosis. Overexpression of the ERBB2 gene is due to increased transcription and gene amplification. Our previous studies have identified a new cis element...

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