Vent® DNA Polymerase

Description

Vent DNA Polymerase is a high-fidelity thermophilic DNA polymerase. The fidelity of Vent DNA Polymerase is 5-15-fold higher than that observed for Taq DNA Polymerase (1,2). This high fidelity derives in part from an integral 3'→5' proofreading exonuclease activity in Vent DNA Polymerase (1,3). Greater than 90% of the polymerase activity remains following a 1 hour incubation at 95°C.



Amplification of Jurkat genomic DNA with Vent DNA Polymerase. Amplicon sizes are indicated below gel. Marker M is the 1 kb DNA Ladder (NEB #N3232 ).

Highlights

  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer and 100 mM MgSO4
  • High-fidelity: 5X greater than Taq
  • High Thermostability: half-life of 6.7 hours at 95°C
  • Value: for routine high-fidelity PCR

Product Source

An E. coli strain that carries the Vent DNA Polymerase gene 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).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Magnesium Sulfate (MgSO4) Solution-20100 mM
ThermoPol® Reaction Buffer Pack-2010X

Advantages and Features

Applications

  • PCR
  • Primer extension

Properties and Usage

Unit Definition

One 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.

Reaction Conditions

1X ThermoPol® Reaction Buffer

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

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
0.1% Triton® X-100
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

No

Molecular Weight

Theoretical: 89000 daltons

5' - 3' Exonuclease

No

3' - 5' Exonuclease

Yes

Strand Displacement

+

Resulting Ends

  • Blunt Ends

Unit Assay Conditions

1X ThermoPol Buffer, 200 µM each dNTP including [3H]-dTTP, 15 nM primed M13 DNA. 

Error Rate

57x10-6bases

Notes

  1. BSA is not provided with this enzyme since its presence is not necessary for most primer extension reactions. However, it is available as NEB #B9001. Acetylated BSA should not be used for primer extension reactions.
  2. The calculated half-life of Vent DNA Polymerase at 95°C is 6.7 hours.
  3. Diluent D is also available. This buffer is recommended for making dilutions of Vent DNA Polymerase.
  4. Additional ThermoPol Reaction Buffer Packs for this product are also available. Each buffer pack contains 4 vials of 10X buffer (1.5 ml each) and 1 vial of 100 mM MgSO4.

References

  1. Mattila, P. et al. (1991). Nucl. Acids Res. 19, 4967-4973.
  2. Eckert, K.A. and Kunkel, T.A. (1991). PCR Methods and Applications. 1, 17-24.
  3. Kong, H.M., Kucera, R.B. and Jack, W.E. (1993). J. Biol. Chem. 268, 1965-1975.
  4. Perler, F. et al. (1992). Proc. Natl. Acad. Sci. USA. 89, 5577.
  5. Belkin, S. and Jannasch, H.W. (1985). Arch. Microbiol. 141, 181-186.

FAQs

  1. Can Vent DNA Polymerase be used in other buffers?
  2. When should VentR DNA Polymerase be used for PCR?
  3. Vent DNA Polymerase and Deep Vent DNA Polymerase produce blunt DNA ends, but my cloning depends on the primer extension product having an adenine single-base 3´ overhang.
  4. I can't get Vent DNA Polymerase to work yet Taq DNA Polymerase works fine.
  5. Are the DNA fragments produced by Vent DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields?
  6. Can I use Vent DNA Polymerase to polish the ends of DNA fragments?
  7. I want to clone primer extension products made with Vent DNA Polymerase or Deep Vent DNA Polymerase. Are some protocols better than others?

Tech Tips

The amount of enzyme in the reaction is critical. Try a final concentration of 0.01-0.02 U/µl Vent DNA Polymerase in the reaction.

Protocols

  1. Guidelines for PCR Optimization for Vent DNA Polymerase
  2. Protocol for a Routine Vent PCR

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Protein Purity (SDS-PAGE):
    The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.