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Klenow (3'-5' exo-)

N-terminal truncated DNA Polymerase I which retains polymerase activity, but no 5´→3´ or 3´→5´exonuclease activity, used for DNA labeling.
 
ENZ-44001-U020 20 µl 204.00 USD
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Klenow (3′-5′ exo-) is a mesophilic DNA polymerase deficient in both proofreading (3′-5′) and nick-translation (5′-3′) nuclease activities, and that displays a moderate strand displacement activity during DNA synthesis. The protein is expressed as a truncated product of the E.coli PolA gene and contains the D355A and E357A mutations.

Product Specification

Alternative Name:Klenow fragment
 
Source:Recombinant E. coli. carrying the Klenow (3'-5' exo-) gene
 
UniProt ID:P00582[324-928]
 
Quantity:20µl
 
Concentration:Unit Concentration: 50 U/µl
Protein Concentration: 5 mg/ml
 
Formulation:Liquid. In 20mM TRIS-HCl, pH 7.5, containing 50% glycerol, 0.1mM EDTA and 1mM DTT.
 
Purity:≥99% (SDS-PAGE)
 
Quality Control:Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme were made in a glycerol (50%) containing Klenow (3′→5′ exo-) storage solution ([Klenow]f= 0.12-0.002 µg/µl) and added to 50 µl reactions containing 4 µg Calf Thymus DNA, 1X Blue Buffer, 4m Ci/ml 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 37°C, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).

SDS-Page (Physical Purity Assessment)
2.0 µl of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µl of a 1:100 dilution of the sample.  Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.

Protein Concentration (OD280) Measurement
A 3.0 µl sample of enzyme was analyzed at OD280using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/ml using an extinction coefficient of 55,450 and molecular weight of 68,067 Daltons. Acceptance for this assay is +/- 5% of reference sample.
 
Appearance:Clear.
 
Specificity:SS Exonuclease: 500U <0.1% released
DS Exonuclease: 500U <0.1% released
Endonuclease: 500U <0.1% converted
E. coli 16S rDNA Contamination 500U <10 copies
 
Specific Activity:10,000 U/mg
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Use/Stability:Stable for at least 1 year after receipt when stored at -20°C.
 
Handling:Minimize repeated freeze/thaw cycles by aliquotting the stock into experimental quantities.
 

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