EpiMark® Hot Start Taq DNA Polymerase

Description

EpiMark Hot Start Taq DNA Polymerase is a mixture of Taq DNA Polymerase and a temperature sensitive, aptamer-based inhibitor. The inhibitor binds reversibly to the enzyme, inhibiting polymerase activity at temperatures below 45°C, but releases the enzyme during normal PCR cycling conditions. This permits assembly of reactions at room temperature. An advantage of the aptamer-based hot start mechanism is that it does not require a separate high temperature incubation step to activate the enzyme. The advanced aptamer-based hot-start activity coupled with the supplied optimized reaction buffer makes the EpiMark Hot Start Taq DNA Polymerase an excellent choice for use on bisulfite-converted DNA.

Taq
DNA polymerase possesses a 5´- 3´ polymerase activity (1,2,3) and a 5´ flap endonuclease activity (4,5).

EpiMark Hot Start Taq DNA Polymerase is supplied with 5X EpiMark Hot Start Taq Reaction Buffer, providing robust amplification for bisulfite-converted DNA and AT-rich amplicons.

Product Source

An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
EpiMark® Hot Start Taq Reaction Buffer Pack-205X

Advantages and Features

Applications

  • PCR from bisulfite-converted DNA

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme that will incorporate 15 nmol dNTP into acid insoluble material in 30 minutes at 75°C.

Reaction Conditions

1X EpiMark® Hot Start Taq Reaction Buffer Pack

1X EpiMark® Hot Start Taq Reaction Buffer Pack:
20 mM Tris-HCl
1.8 mM MgCl2
22 mM KCl
0.06% IGEPAL® CA-630
0.05% Tween® 20
22 mM NH4Cl
pH 8.9 @ 25°C

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
0.5% Tween® 20
0.5% IGEPAL® CA-630
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

No

Molecular Weight

Theoretical: 94000 daltons

5' - 3' Exonuclease

Yes

3' - 5' Exonuclease

No

Resulting Ends

  • Single-base 3´ Overhangs

Unit Assay Conditions

1X ThermoPol® Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 15 nM primed M13 DNA. 

References

  1. Chien, A., Edgar, D.B. and Trela, J.M. (1976). J. Bact. . 127, 1550-1557.
  2. Kaledin, A.S., Slyusarenko, A.G. and Gorodetskii, S.I. (1980). Biokhimiya . 45, 644-651.
  3. Lawyer, F.C. et al. (1993). PCR Methods And Appl.. 2, 275-287.
  4. Longley, M.J., Bennett, S.E. and Mosbaugh D.W. (1990). Nucleic Acids Res. . 18, 7317-7322.
  5. Lyamichev, V., Brow, M.A. and Dahlberg, J.E. (1993). Science. 260, 778-783.
  6. Saiki R.K. et al. (1985). Science. 230, 1350-1354.
  7. Powell, L.M. et al. (1987). Cell. 50, 831-840.
  8. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques . 15, 372-374.

FAQs

  1. Does EpiMark® Hot Start Taq DNA Polymerase require an extended initial incubation to activate the polymerase?
  2. How has this product been optimized for use with bisulfite-converted DNA?
  3. Can EpiMark® Hot Start Taq DNA Polymerase be used in any other reaction buffers?
  4. What type of PCR ends are produced with EpiMark® Hot Start Taq DNA Polymerase?

Protocols

  1. EpiMark® Hot Start Taq DNA Polymerase Guidelines for PCR (M0490)

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

Interactive Tools

NEB Publications

  • Estève P., Zhang G., Ponnaluri V., Kanneganti (2015). Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates Nucleic Acids Res. PubMedID: 26553800, DOI: 10.1093/nar/gkv1162

Citations

  • Chernov AV., Reyes L., Peterson S., Strongin AY. (2015). Depletion of CG-Specific Methylation in Mycoplasma hyorhinis Genomic DNA after Host Cell Invasion PLoS One. 10, e0142529. PubMedID: 26544880, DOI: 10.1371/journal.pone.0142529
  • Page A., Paoli P., Salvador E., White S., French J., Mann J. (2015). Hepatic Stellate Cell Transdifferentiation Involves Genome-Wide Remodeling of the DNA Methylation Landscape J Hepatol. PubMedID: 26632634, DOI: 10.1016/j.jhep.2015.11.024

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.
  • Inhibition of Primer Extension (Hot Start, Radioactivity Incorporation):
    The hot start polymerase is compared to the polymerase in non-hot start conditions using primer extension; inhibition is assessed by comparing radioactive incorporation.
  • PCR Amplification (Hot Start):
    The polymerase is tested in a hot start polymerase chain reaction (PCR) using Lambda DNA as the control template, specific primers and human genomic DNA, resulting in an increase in yield of the expected Lambda product and a decrease in non-specific genomic bands when compared to a non-hot start control reaction.
  • Single Stranded DNase Activity (FAM Labeled Oligo):
    The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide. The percent degradation is determined by capillary electrophoresis.

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.