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VentR® (exo-) DNA Polymerase |
 | | | | VentR® (exo-) DNA Polymerase | | | |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer and MgSO4
- High-Fidelity: 1.5X greater than Taq
- High Thermostability: half-life of 6.7 hours at 95°C
- Value: for routine high-fidelity PCR
Description:
VentR (exo-) DNA Polymerase has been genetically engineered to eliminate the 3´→ 5´ proofreading exonuclease activity associated with VentR DNA Polymerase (3). This is the preferred form for high-temperature dideoxy sequencing reactions and for high yield primer extension reactions. The fidelity of polymerization by this form is reduced to a level about 2-fold higher than that of Taq DNA Polymerase (1,2).
Source:
An E. coli strain that carries the Vent (D141A / E143A) DNA Polymerase gene, a genetically engineered form of the native DNA polymerase from Thermococcus litoralis (4). The native organism is capable of growth at up to 98°C and was isolated from a submarine thermal vent (5).
Applications:- Primer extension
- Thermal cycle sequencing
- High temperature dideoxy-sequencing
- PCR
Reagents Supplied:
MgSO4 (100 mM)
ThermoPol Reaction Buffer (10X)
Enzyme Properties
Polymerase Properties | Thermophilic Polymerase Characteristics
3´ to 5´ Exonuclease: No
5´ to 3´ Exonuclease: No
Strand Displacement: Yes
Error Rate: 190
x10-6 bases
Heat Inactivation:
No
Molecular Weight:
Theoretical: 89,000 daltons
Specific Activity:
28,000 units/mg
Reaction & Storage Conditions
Reaction Conditions:
1X ThermoPol Reaction Buffer
Supplemented with 200 μM dNTPs (not included) and 200 μg/ml activated calf thymus DNA
1X ThermoPol Reaction Buffer:
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1 % Triton X-100
pH 8.8 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in a total reaction volume of 50 μl in 30 minutes at 75°C in 1X ThermoPol Reaction Buffer with 200 µM dNTPs including [3H]-dTTP and 200 µg/ml activated Calf Thymus DNA.
Unit Assay Conditions: 1X ThermoPol Reaction Buffer, 200 µM each dNTP including [3H]-dTTP, 200 µg/ml activated calf thymus DNA.
Concentration:
2,000 units/ml
Storage Conditions:
10 mM Tris-HCl
100 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
General notes:- Vent Diluent is also available (NEB #B8004S). This buffer is recommended for making dilutions of VentR® (exo-) Polymerase.
- Additional buffer packs for this product are also available (NEB #B9004S). Each buffer pack contains 4 vials of 10X buffer (1.5 mls each), and 1 vial of 100 mM MgS04.
Usage notes:- BSA is not provided with this enzyme since its presence is not necessary for most primer extension reactions. However, it is available free of charge, if requested when placing an order. Acetylated BSA should not be used for primer extension reactions.
FAQs
- Can Vent (exo-) DNA Polymerase be used in other buffers?
- Are the DNA fragments produced by Vent (exo-) DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields?
- I can't get Vent (exo-) DNA Polymerase to work yet Taq DNA Polymerase works fine.
Protocols
Protocols for VentR® (exo-) DNA Polymerase
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
Purified free of contaminating endonucleases and exonucleases.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of VentR® (exo-) DNA Polymerase with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 75ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 200 units of VentR® (exo-) DNA Polymerase with 1 μg of
ΦX174 DNA for 4 hours at 75ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
3' → 5' Exonuclease Activity:
Incubation of a 20 µl reaction in ThermoPol Reaction Buffer containing a minimum of 20 units of VentR (exo-) DNA Polymerase with 10 nM fluorescent internally labeled oligonucleotide for 30 minutes at either 37°C or 75°C yields no detectable 3´→ 5´ degradation as determined by capillary electrophoresis.
Physical Purity:
Purified to > 95% homogeneity as determined by SDS-PAGE analysis using Coomassie Blue detection.
References
- Mattila, P. et al. (1991) Nucl. Acids Res., 19, 4967-4973.
- Eckert, K.A. and Kunkel, T.A. (1991) PCR Methods and Applications, 1, 17-24.
- Kong, H.M. et al. (1993) J. Biol. Chem., 268, 1965-1975.
- Perler, F.B. et al. (1992) Proc. Natl. Acad. Sci. USA, 89, 5577-5581.
- Belkin, S. and Jannasch, H.W. (1985) Arch. Microbiol., 141, 181-186.
Reagents Sold Separately
ThermoPol Reaction Buffer
Companion Products
BSA
Deoxynucleotide Solution Mix
Deoxynucleotide Solution Set
Diluent D
Magnesium Sulfate (MgSO4) Solution
ThermoPol DF (Detergent-free) Reaction Buffer
ThermoPol II (Mg-free) Reaction Buffer Pack
ThermoPol Reaction Buffer
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,322,785
New England Biolabs, Inc.: U.S. Patent No. 5,210,036
New England Biolabs, Inc.: U.S. Patent No. 5,352,778
New England Biolabs, Inc.: U.S. Patent No. 5,500,363
Licenses/Patents/Disclaimers:
Some applications in which this product can be used may be covered by patents issued and applicable in the United States and certain other countries. Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a patent license depending upon the particular application in which the product is used.
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New
England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201 |
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