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Maxima™ Hot Start Taq DNA Polymerase

#EP0601 Supplied with: 10X Hot Start PCR Buffer 25 mM MgCl2	100 u (5 u/µl) 0.6 ml 0.6 ml
#EP0602 Supplied with: 10X Hot Start PCR Buffer 25 mM MgCl2	500 u (5 u/µl) 2x1.25 ml 2x1.25 ml
#EP0603 Supplied with: 10X Hot Start PCR Buffer 25 mM MgCl2	5x500 u (5 u/µl) 10x1.25 ml 10x1.25 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0601, #EP0602, #EP0603 MSDS (English) MSDS (English-USA) MSDS (German) Flyer
Protocol for PCR with Hot Start Taq DNA Polymerase

 

Features

• High yield amplification of complex templates.
• Short 4 min activation time.
• High PCR specificity - reduced effects of mispriming and primer-dimer formation.
• Enhanced PCR sensitivity.
• Convenient room temperature PCR set-up.
• Generates PCR products with 3'-dA overhangs.

TrueStart™ and Hot Start  Taq DNA Polymerases, flyer in pdf, 182 KB

Description
Maxima™ Hot Start Taq DNA Polymerase is designed to enhance the specificity, sensitivity and yield of DNA amplification (1-4). In addition, the enzyme provides the convenience of reaction set-up at room temperature. Maxima™ Hot Start Taq DNA Polymerase is a recombinant Taq DNA polymerase which has been chemically modified by the addition of heat-labile blocking groups to its amino acid residues. The enzyme is inactive at room temperature, avoiding extension of non-specifically annealed primers or primer dimers and providing higher specificity of DNA amplification (see Fig.2). The functional activity of the enzyme is restored during a short 4-minute incubation at 95°C. The activated enzyme maintains the same functionality as Taq DNA polymerase: catalyzes 5'=>3' synthesis of DNA, has no detectable 3'=>5' proofreading exonuclease activity, but possesses low 5'=>3' exonuclease activity. It exhibits deoxynucleotidyl transferase activity, which frequently results in the addition of extra adenines at the 3'-end of PCR products. Before activation, the two activities are not detectable.

Applications

• High yield amplification of targets up to 3 kb.
• Hot start PCR.
• RT-PCR.
• Highly specific amplification of complex genomic and cDNA templates.
• Amplification of low copy DNA targets.
• Real-time PCR.
• Multiplex PCR.
• Generation of PCR products for TA cloning.

Molecular Weight
94 kDa monomer.

Concentration
5 u/µl

Quality Control
The absence of endodeoxyribonucleases, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested in hot-start PCR.

Definition of Activity Unit
One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 70°C.
Enzyme activity is assayed in the following mixture: 67 mM Tris-HCl (pH 8.8 at 25°C), 6.7 mM MgCl₂, 1 mM 2-mercaptoethanol, 50 mM NaCl, 0.1 mg/ml BSA, 0.75 mM activated calf thymus DNA, 0.2 mM of each dNTP, 0.4 MBq/ml [³H]-dTTP.

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 9.0), 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, 0.5% (v/v) Tween 20 and 50% (v/v) glycerol.

10X Hot Start PCR Buffer
200 mM Tris-HCl (pH 8.3 at 25°C), 200 mM KCl, 50 mM (NH₄)₂SO₄.

Inhibition and Inactivation
Inactivated by phenol/chloroform extraction.

Figure 1. Maxima™ Hot Start Taq DNA Polymerase is inactive at low temperatures.
Enzyme activity assays were performed at 25°C in one hour intervals with Maxima™ Hot Start Taq DNA Polymerase and Taq DNA polymerase.

Figure 2. Specific amplification of a 2 kb fragment from human beta-globine gene using Maxima™ Hot Start Taq DNA Polymerase.
50 ng of human genomic DNA was used as a template for amplification of a 2 kb fragment of the beta-globine gene using hot start Taq DNA polymerases from different vendors in 35 cycles on the GeneAmp® 9700 PCR System.
M - GeneRuler™ 100 bp Plus DNA Ladder
1 -  Maxima™ Hot Start Taq DNA Polymerase, Fermentas
2 -  Hot start Taq DNA polymerase (chemical modification), Vendor A
3 -  Hot start Taq DNA polymerase (temperature-dependent inhibitor), Vendor B

Related Products

PyroStart™ Fast PCR Master Mix (2X) dNTP Mixes dNTP Set Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP CloneJET™ PCR Cloning Kit InsTAclone™ PCR Cloning Kit First Strand cDNA Synthesis Kit RevertAid™ First Strand cDNA Synthesis Kits M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase FastRuler™ DNA Ladders, ready-to-use O'RangeRuler™ DNA Ladders, ready-to-use GeneRuler™ DNA Ladders T4 DNA Polymerase Genomic DNA Purification Kit DNA Gel Extraction Kit Agarase Water, nuclease-free

References

1. D’Aquila, R.T., et al., Maximizing sensitivity and specificity of PCR by preamplification heating, Nucleic Acids Res., 19, 3749, 1991.
2. Kellogg, D.E., et al., TaqStart antibody: "Hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase, BioTechniques, 16, 1134-1137, 1994.
3. Horton, R.M., et al., AmpliGrease: "Hot Start" PCR using petroleum jelly, BioTechniques, 16, 42-43, 1994.
4. Dang, C. and Jayasena, S.D., Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR, J. Mol. Biol., 264, 268-278, 1996.
5. Weyant, R.S., et al., Effect of ionic and nonionic detergents on the Taq polymerase, Biotechniques, 9, 309-308, 1990.

Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights  (such as 5' Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. For complete license disclaimer, see.

Updated kovo 18, 2008 09:40