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商品編號


P7260L



品牌


Enzymatics



公司


Enzymatics,inc



公司分類(lèi)


Ultrapure Polymerases



Size

3,500 U

商品信息

T7 DNA Polymerase


Product Description

T7 DNA Polymerase is the mesophilic, highly processive, replicative DNA polymerase from bacteriophage T7 that is respons
IBL
e for the rapid and accurate replication of the virus’ genome during its infection cycle. T7 DNA Polymerase is a two subunit protein, consisting of a polymerase domain (gene 5 from the T7 bacteriophage) and a processivity factor (
E. coli
?trxA gene thioredoxin) (1,2). The enzyme possesses a powerful (3’→5′) nuclease activity that acts on both single and double stranded DNA and appears to be respons
IBL
e for the high fidelity of this enzyme and prevents strand displacement synthesis (3,4,5).

Source of Protein
A recombinant?
E. coli
?strain carrying the bacteriophage T7 gene 5.

Supplied in
50 mM KPO
4
0.1 mM EDTA
1.0 mM DTT
50% glycerol
pH 7.0 @ 25°C

Supplied With
B7260 (10X T7 DNA Polymerase Buffer)

10X T7 DNA Polymerase Buffer (B7260)
400 mM Tris-HCl
200 mM MgCl
2
500 mM NaCl
pH 7.5 @ 25°C

Unit Definition
1 unit is defined as the amount of polymerase required to convert 10 nmol of total 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 1X reaction buffer and added to 50 ?L reactions containing Calf Thymus DNA, 1X T7 DNA Polymerase Unit Cheracterization Buffer (20mM Tris-HCl, 100mM KCl, 6mM MgCl
2
, 0.1mM EDTA, 5mM β-mercaptoethanol),?
3
H-dTTP and 150 ?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 157,180 and molecular weight of 92,140 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 greater than 50% 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 greater than 50% release of TCA-soluble counts.

Double-Stranded Endonuclease Activity
A 50 ?L reaction containing 0.5 ?g of pBR322 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.


View PDF Poster Instructions FAQ


Product Information



T7 DNA Polymerase


Part Number
P7260L
Price
$363
Concentration
10,000 U/ml
Unit Size
3,500 U


SDS

Available on request








Product Specification*


Storage Temperature
-25 to -15°C


Test

Specification


Purity (SDS-PAGE)
>99%
Specific Activity
13,333 U/mg
SS Exonuclease
Functional
DS Exonuclease
Functional
DS Endonuclease
100 U = No conversion
E.coli
DNA Contamination
100 U <10 copies





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

Usage Instructions

The following workflow can be applied to either the production of a second DNA strand following first-strand synthesis off an RNA template, or to a site-directed mutagenesis experiment where the mutagenic primer is extended such that the entire template strand is copied.

In a sterile reaction vessel, combine the following components:




Amount

Description

Final Concentration



20 ?L

Pre-annealed primer/template

20 nmol complex



4 ?L

10X T7 DNA Pol. Buffer

1X



0.5 ?L

25 mM dNTP Solution (N2050L)

300 ?M



2 ?L

T7 DNA Polymerase (P7260L)

500 U/mL



2 ?L

T4 DNA Ligase (L6030-LC-L)

2,400 U/mL



10 ?L

Type I Water

N/A



40 ?L

Total Volume




?

Incubate 60 min at 37°C. Incubate 10 min at 70°C to stop the reaction.

Notes

This enzyme is not suitable for DNA sequencing.

References


Grippo, P. et al. (1971) J. Biol. Chem. 246, 6867-6873.

Modrich, P. et al. (1975) J. Biol. Chem. 250, 5515-5522.

Adler, S. et al. (1979) J. Biol. Chem. 254, 11605-11614

Hori, K., et al. (1979) J. Biol. Chem. 254, 11598-11604.

Lechner, R. L. et al. (1983) J. Biol. Chem. 258, 11185-11196.




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.