TopTaq DNA Polymerase Enzyme

TopTaq DNA Polymerase contains three binding proteins: one binding protein binds to double-stranded DNA template, preventing polymerase activity at room temperature, and the other two binding proteins bind primers, preventing primer-dimer formation. Together, these binding proteins reduce non-specific amplification. The blocking proteins are released from primers and templates during the initial denaturation. This double-blocking method has higher efficiency and specificity than antibody-based or chemically-modified hot start PCR. Template-independent "A" can be generated at the 3' end of the PCR product. PCR products can be directly cloned into T-vectors. This kit does not use Taq antibodies, reducing the risk of mammalian DNA contamination, and this kit does not use chemical modification, so a long denaturing step is not required. Genomic DNA fragments can be amplified up to 15 kb. This kit does not contain 2.5 mM dNTPs.

Contents:

Component	250 U	500 U	3 kU
TopTaq DNA Polymerase	250 U	500 U	6 × 500 U
10X TopTaq Buffer	1.2 ml	2 × 1.2 ml	12 × 1.2 ml
10X GC Enhancer	200 µl	400 µl	1 ml
6X DNA Loading Buffer	500 µl	1 ml	2 × 1 ml

Target	TopTaq DNA Polymerase
Tested Applications	PCR
Conjugation	Unconjugated
Purity	> 99% (SDS-PAGE)
Quality Control	Assayed for amplication efficiency to amplify the p53 gene from 10 ng of human genomic DNA.
Storage	Store at -20°C for up to 2 years. Avoid repeated freeze/thaw cycles.
Buffer	TopTaq DNA Polymerase: 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 50% glycerol, stabilizers. 10X TopTaq Buffer: 500 mM Tris-HCl (pH 9.0), 200 mM (NH4)2SO4, 20 mM MgSO4, other proprietary ingredients.
Biological Activity	One unit of TopTaq DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 minutes at 74 °C.
Endotoxin Level	Functional absence of double and single stranded endonuclease activity.
Concentration	2.5 U/µl
Availability	Shipped within 10-20 working days.
Note	This product is for research use only. Not for human consumption, cosmetic, therapeutic or diagnostic use.
Directions for use	Reaction Components: Component Volume Final Concentration Template Variable as required Forward Primer (10 µM) 1 µl 0.2 µM Reverse Primer (10 µM) 1 µl 0.2 µM 10X TopTaq Buffer 5 µl 1X 2.5 mM dNTPs 4 µl 0.2 mM TopTaq DNA Polymerase 0.5-1 µl 1.25-2.5 U Nuclease-free H2O Variable N/A Total Volume 50 µl N/A Thermal Cycling Conditions: Number of Cycles Temperature Time 1 cycle 94 °C 2-5 min 30-35 cycles 94 °C 30 seconds 50-60 °C 30 seconds 72 °C 1-2 kb/min 1 cycle 72 °C 5-10 min Notes: For GC/AT-rich or complex templates, it is recommended to add GC Enhancer to the PCR reaction mixture. The suggested working concentration range for the 10X GC Enhancer provided in this kit is 0.5-5X. A final concentration of 2 mM MgSO4 is sufficient to amplify most targets. Some targets may require a higher concentration of Mg2+ For optimal results, we recommend using a 100 mM MgSO4 stock solution to prepare a titration from 2 mM to 4 mM (final concentration) in 0.25 mM increments. 0.5 µl (2.5 U) of enzyme is sufficient for a reaction vlume of 50 µl. For increased amplification, up to 1 µl (5 U) of enzyme can be used.

Research Articles on TopTaq DNA Polymerase

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