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商品編號


P7140-HC-L



品牌


Enzymatics



公司


Enzymatics,inc



公司分類


Ultrapure Polymerases



Size

100,000 U

商品信息

Manta 1.0 DNA Polymerase (High Concentration)


Product Description

Manta 1.0 DNA Polymerase (exo-) is a
Thermo
philic DNA polymerase deficient in both proofre
ADI
ng (3′→ 5′) and nick-translation (5′→ 3′) nuclease activities. The protein was originally characterized and its crystal structure solved by Lorena Beese (1).

Source of Protein

A recombinant?
E. coli
?strain carrying the Manta 1.0 DNA Polymerase (exo-) polymerase gene.

Supplied in

10 mM Tris-HCl

50 mM KCl
1.0 mM DTT
0.1 mM EDTA
0.1% Triton X-100
50% glycerol
pH 7.5 @ 25°C

Supplied With
B7140 (10X PCR Buffer II)

10X PCR Buffer ll (B7140):
200 mM Tris-HCl

100 mM (NH
4
)
2
SO
4
100 mM KCl
20 mM MgSO
4
1.0% Triton X-100
pH 8.8 @ 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 65°C.



Quality Control Analysis

Unit Characterization Assay
Unit activity was measured using a 2-fold serial dilution method. Dilutions of enzyme batch were made in 1X reaction buffer and added to 50 ?L reactions containing Calf Thymus DNA, 1X PCR Buffer II,?
3
H-dTTP and 100 ?M dNTPs. Reactions were incubated 10 minutes at 65°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 52,770 and molecular weight of 66,215 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 5.0% 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 pBR322 DNA and 5 ?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.

?


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Product Information



Manta 1.0 DNA Polymerase (High Concentration)


Part Number
P7140-HC-L
Price
$363.00
Concentration
400,000 U/ml
Unit Size
100,000 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
Specific Activity
400,000 U/mg
SS Exonuclease
4000 U <5.0% released
DS Exonuclease
4000 U <1.0% released
DS Endonuclease
4000 U = no conversion
E.coli
DNA Contamination
4000 U <10 copies





* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

Notes

Specific activity measured under above conditions is approximately 3X higher than cited by Beese et al. when using activated calf thymus DNA as a substrate.

References


Kiefer, et al. Structure 15 January 1997. 5, 95-108.




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.