商品编号


P7010-HC-L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


Ultrapure Polymerases



Size

10,000 U

商品信息

Klenow (3′→ 5′ exo-) (High Concentration)


Product Description

Klenow?(3′→ 5′ exo-) is a mesophilic?DNA polymerasedeficient in both proofre
ADI
ng (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.

Supplied in?
20 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
50% glycerol
pH 7.5 @ 25°C

Supplied With
B0110 (10X Blue Buffer)

10X Blue Buffer (B0110)

500mM NaCl
100 mM Tris-HCl
100 mM MgCl
2
10 mM DTT
pH 7.9 @ 25°C

Unit Definition
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.



Quality Control Analysis

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 ?L reactions containing Calf Thymus DNA, 1X Blue Buffer,
3
H-dTTP and 100 ?M 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 (OD
280
) Measurement
A 2.0 ?L sample of enzyme was analyzed at OD
280
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 ?L 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 ?L 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.



Contamination Tests

Single-Stranded Exonuclease Activity
A 50 ?L reaction containing 10,000 cpm of a r
ADI
olabeled single-stranded DNA substrate and 10 ?L 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 ?L reaction containing 5,000 cpm of a r
ADI
olabeled double-stranded DNA substrate and 10 ?L 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 ?L reaction containing 0.5 ?g of pENZuC DNA and 10 ?L of enzyme solution incubated for 4 hours at 37°C resulted in no visually discern
IBL
e conversion to nicked circular DNA as determined by agarose gel electrophoresis.

E.coli
16S rDNA Contamination Test
Replicate 5 ?L 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 (C
t
) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control C
t
values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.

UDG Contamination Test
A 50 ?L reaction containing tritiated Uracil DNA and 10 ?L 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.


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Product Information



Klenow (3′→ 5′ exo-) (High Concentration)


Part Number
P7010-HC-L
Price
$393
Concentration
50,000 U/ml
Unit Size
10,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
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 admi
NIST
ration to humans or animals. SDS sheets relevant to this product are available upon request.