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.
Source of Protein
A recombinant E. coli strain carrying the Klenow (3′→5′ exo-) gene.
20 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
pH 7.5 @ 25°C
B0110 (10X Blue Buffer)
10X Blue Buffer (B0110)
100 mM Tris-HCl
100 mM MgCl2
10 mM DTT
pH 7.9 @ 25°C
1 unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material
in 30 minutes at 37°C.
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 and added to 50 µL reactions containing Calf Thymus DNA, 1X Blue Buffer, 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).
Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using 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 55,330 and molecular weight of 68,100 Daltons.
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.
Single-Stranded Exonuclease Activity
A 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 10.0% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pENZuC DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate 5 µL samples of enzyme solution were denatured and screened in a TaqMan qPCR assay for the presence of contaminating E.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
UDG Contamination Test
A 50 µL reaction containing tritiated Uracil DNA and 10 µL enzyme solution incubated for 60 minutes at 37°C under standard UDG unit characterization conditions resulted in the measurement of less than 20 U/mL UDG activity as determined by liquid scintillation analysis.
|Klenow (3′→ 5′ exo-) (Low Concentration)|
|Unit Size||10,000 U|
|SDS||Available on request|
|Storage Temperature||-25 to -15°C|
|Specific Activity||10,000 U/mg|
|SS Exonuclease||500 U <10.0% released|
|DS Exonuclease||500 U <1.0% released|
|DS Endonuclease||500 U = no conversion|
|E.coli DNA Contamination||500 U <10 copies|
|UDG Activity||< 20 U/mL activity|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.
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