商品编号


P7250L



品牌


Enzymatics



公司


Enzymatics,inc



公司分类


Ultrapure Polymerases



Size

10,000 U

商品信息

Taq-B DNA Polymerase


Product Description

Taq-B DNA Polymerase is a thermally stable, processive, 5′→ 3′ DNA polymerase. The 94 kDa protein possesses an inherent 5′→ 3′ nick-translation moiety and lacks a 3′→ 5′ proofre
ADI
ng function.

Source of Protein

A recombinant?
E. coli
?strain carrying the Taq DNA polymerase gene from the
Thermo
philic organism?
Thermus Aquaticus
?YT-1.

Supplied in

20 mM Tris-HCl

100 mM NaCl
1.0 mM DTT
0.1 mM EDTA
St
ABI
lizer
50% glycerol
pH 7.5 @ 25°C

Supplied With
B7030 (10X PCR Buffer I)

10X PCR Buffer l (B7030)
100 mM Tris-HCl?

500 mM KCl
15 mM MgCl
2
pH 8.3 @ 25°C

Unit Definition

1 unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.



Quality Control Analysis

Unit Characterization Assay
Specific 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, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0mM MgCl
2
, 1 mM DTT,?
3
H-dTTP and 100 ?M dNTPs. Reactions were incubated 10 minutes at 75°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 110,380 and molecular weight of 93,910 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 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 5uL 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



Taq-B DNA Polymerase


Part Number
P7250L
Price
$363
Concentration
5,000 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
74,625 U/mg
SS Exonuclease
50 U <5.0% released
DS Exonuclease
50 U <1.0% released
DS Endonuclease
50 U = No conversion
E.coli
DNA Contamination
50 U <10 copies





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

Usage Instructions

Typical 50 ?L Reaction

On ice, prepare each of following master mixes, combine, and place in heated (to 94°C) thermal cycler:

2X DNA/Oligonucleotide Master Mix:
1.0 ?L 10 mM dNTPs
1.0 ?L 10 ?M Forward Primer
1.0 ?L 10 ?M Reverse Primer
1.0 ?L 500 ng/?L genomic DNA
21 ?L Type I Water

2X Enzyme/Buffer Master Mix:
5.0 ?L 10X PCR Buffer I
0.2 ?L 5 U/?L Taq DNA Polymerase
19.8 ?L Type I Water

General Cycling Conditions

94°C?????? 3 minutes Initial Denaturation

25 Cycles:

94°C?????? 30 seconds???????????? Denaturation
55°C?????? 30 seconds???????????? Annealing
68°C?????? 30 seconds ??????????? 500 bp extension
68°C?????? 5 minutes ???????????????Final Extension

Notes

Taq DNA Polymerase is the original and most commonly used PCR enzyme. Taq excels at amplifying shorter (<5 kb) sequences from low-complexity template sources and produces robust yields with little or no optimization of reaction conditions. Consider the following guidelines when designing PCR strategies using Taq DNA Polymerase.


DNA Template:?
Although extensive purification of PCR templates is typically not necessary, care should be taken with crude or partially purified DNA sources as handling and chemical agents can adversely affect the PCR process. Exposure to short-wave UV light or other DNA damaging agents should be avoided, as should high ionic strength, detergents such as SDS, lo
ADI
ng dyes and phenol. In order to prevent contamination from previous PCR reactions, consider setting up reactions in a positive-pressure hood and with aerosol barrier pipet tips. In a typical 25 cycle PCR, 104 copies of target sequence will yield reproduc
IBL
e amplification product. This corresponds to roughly 0.1-1 ng/ml (final concentration) of plasmid DNA, and 1-10 ?g/ml of genomic DNA. The use of lower DNA concentrations typically produces less non-specific product, while higher concentrations can allow for fewer cycles and lower mutation rates.

Primer Design:
IIdeally, oligonucleotide primers are 15-30 bases in length, nearly 50% G+C, and have equal (+/- 3°C) annealing temperatures. The use of software to detect self-complementary or hairpin-prone regions is advised and is offered as a service by some synthesis providers. Note that although the 5′-terminus of the primer may contain untemplated sequence, the 3&prime end must match perfectly. Typical oligonucleotide concentration in the reaction is 0.1-0.5 ?M.

Magnesium:
Magnesium is a critical component of the PCR reaction though its concentration can be modulated to promote various effects. Generally, 1.5-2.0 mM Mg2+ is targeted, but higher concentrations (up to 5 mM) may be used to stimulate the yield of reactions at the expense of fidelity. The converse is also true – lower magnesium concentrations will promote higher- fidelity products with a lower overall amplification yield. Note that certain reaction components, in particular template DNA and oligonucleotides, may contribute chelating agents to the reaction which could lower the effective magnesium concentration and starve the reaction.

dNTPs:
Generally, a final concentration of 100-200 ?M dNTPs is employed, though higher concentrations may stimulate yields (particularly with longer targets) and lower may offer increases in fidelity. Taq DNA Polymerase can also incorporate and read through deoxy Uridine and Inosine, two analogs used in certain applications.

Taq Polymerase:
1 unit/50 ?L reaction (20 U/mL) is typical, though additional enzyme may be added to stimulate yields. Taq DNA Polymerase extends a DNA template at approximately 2000 nucleotides/minute, so it is recommended that 45-60 seconds of extension time be provided per cycle. Appropriate extension temperatures range from 66-72°C.


Legal Disclaimer

Patents
Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application, therefore it is the sole responsibility of users of this product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used.



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.