LongAmp® Hot Start Taq 2X Master Mix

Description

LongAmp Hot Start Taq 2X Master Mix contains a unique blend of aptamer-based Hot Start Taq and Deep VentR™ DNA Polymerases. The aptamer-based 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 PCR reactions at room temperature. The LongAmp® Hot Start Taq 2X Master Mix does not require a separate high temperature incubation step to activate the enzyme. The 3´→5´ exonuclease activity of Deep VentR DNA Polymerase increases the fidelity and robust amplification of Hot Start Taq DNA Polymerase (1). LongAmp® Hot Start Taq DNA Polymerase offers two-fold higher fidelity than Hot Start Taq DNA Polymerase alone. The convenient master mix formulation is supplied at a 2X concentration and contains dNTPs and Mg++, requiring only the addition of primers and DNA template for robust amplification. A wide range of PCR products can be generated; up to 30 kb from lambda or human genomic DNA.

Amplification of a 8 kb amplicon from varying amounts of Jurkat genomic DNA using LongAmp Hot Start Taq 2X Master Mix. Starting template amounts are indicated below the gel. Marker M is the NEB 2-Log DNA Ladder (NEB #N3200 ).

Product Source

An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1 and an E. coli strain that carries the Deep VentR DNA Polymerase gene from Pyrococcus species GB-D.

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.

Unit Assay Conditions:
1X ThermoPol™ Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 200 µg/ml activated Calf Thymus DNA.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
LongAmp® Taq Reaction Buffer Pack-205X

Advantages and Features

Applications

  • High-specificity Long Range PCR 
  • High-throughput PCR
  • Colony PCR

Properties and Usage

1X Master Mix Composition

60 mM Tris-SO4
20 mM (NH4)2SO4
2 mM MgSO4
3% Glycerol
0.06% IGEPAL® CA-630
0.05% Tween® 20
0.3 mM dNTPs
125 units/ml LongAmp® Hot Start Taq DNA Polymerase
pH 9.1@25°C

Heat Inactivation

No

Unit Assay Conditions

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

Notes

  1. Product specifications for individual components in the LongAmp Hot Start Taq 2X Master Mix are available separately.

References

  1. Barnes, W.M. (1994). Proc. Natl. Acad. Sci. USA. 91, 1350-1354.
  2. Saiki R.K. et al. (1985). Science. 230, 1350-1354.
  3. Powell, L.M. et al (1987). Cell. 50, 831-840.
  4. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
  5. Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res.. 18, 7465.

FAQs

  1. What is the fidelity of the LongAmp™ Hot Start Taq 2X Master Mix compared to Taq DNA Polymerase?
  2. Can the extension step be carried out at 72°C when using LongAmp™ Hot Start Taq 2X Master Mix?
  3. What is the extension rate when using LongAmp™ Hot Start Taq 2X Master Mix?
  4. What type of DNA ends result from a primer extension reaction or a PCR using LongAmp™ Hot Start Taq 2X Master Mix?
  5. Why is the product a smear when visualized on an agarose gel?
  6. Can LongAmp™ Hot Start Taq 2X Master Mix be used to amplify GC-rich amplicons?

Tech Tips

Using an extension temperature of 65°C is recommended in LongAmp Hot Start Taq reactions.

Protocols

  1. Protocol for LongAmp™ Hot Start Taq 2X Master Mix

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

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.
  • 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.
  • PCR Amplification (Master Mix):
    The polymerase master mix is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.

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.