Phusion Hot Start II High-Fidelity DNA Polymerase

The most accurate hot start DNA polymerase on the market featuring extreme specificity and improved performance.
Thermo Scientific Phusion Hot Start II High Fidelity DNA Polymerase is highly accurate hot start DNA polymerase. It combines the Phusion DNA Polymerase and a reversibly bound, specific Affibody ligand. Affibody inhibits the DNA polymerase activity at room temperature and thus prevents the amplification of nonspecific products.

The Affibody ligand also inhibits the 3´→5´ exonuclease activity of the polymerase, preventing degradation of primers and template DNA during reaction set up. At polymerization temperatures, the ligand is released, rendering the polymerase fully active. Phusion Hot Start II DNA Polymerase does not require a separate activation step in the PCR protocol as it is immediately reactivated at high temperatures.

Phusion Green Hot Start II High-Fidelity DNA Polymerase is a combination of Phusion Hot Start II DNA Polymerase and 5X Phusion Green Buffers. The buffers includes a density reagent and two tracking dyes for direct loading of PCR products on a gel. The colored buffer does not interfere with Phusion Hot Start II DNA Polymerase performance and is compatible with downstream applications such as DNA sequencing, ligation and restriction digestion.

Phusion Hot Start II High-Fidelity PCR Master Mixes are convenient 2X mixes designed to minimize the number of pipetting steps. The master mixes contain Phusion Hot Start II DNA Polymerase, nucleotides and optimized reaction buffer including MgCl2. Only template and primers need to be added to the PCR reaction.
Phusion PolymerasesDID YOU KNOW?

The optimal annealing temperature for Phusion DNA Polymerases may differ significantly from that of Taq-based polymerases.

For optimal results start by accurately calculating your Tm with our Tm calculator.


  • Reaction set up at room temperature
  • No non-specific amplification and primer degradation during reaction set up
  • Zero-time reactivation due to unique hot start technology
  • High fidelity (52X Taq)
  • Fast PCR due to short extension times (15-30 s/kb)
  • Robust reactions, minimal optimization needed
  • Increased product yields with minimal enzyme amounts
  • Direct loading on gels with Green Buffer


  • High-fidelity PCR
  • High throughput
  • Difficult (GC-rich) templates
  • Template generation for sequencing
  • Multiplex PCR
  • Long-range PCR
  • Cloning
  • Mutagenesis†
  • Microarray

Phusion Site-Directed Mutagenesis Kit also available.


Phusion and Phusion Green Hot Start II High-Fidelity DNA Polymerase

  • Phusion Hot Start II High-Fidelity DNA Polymerase (2 U/µL)
  • 5X Phusion HF or Phusion Green HF buffer (provides 1.5 mM MgCl2 in the final 1X concentration)
  • 5X Phusion GC or Phusion Green GC buffer (provides 1.5 mM MgCl2 in the final 1X concentration)
  • DMSO
  • 50 mM MgCl2 solution

Phusion and Phusion Green Hot Start II High-Fidelity PCR Master Mix

  • 2X Phusion or Phusion Green Hot Start II High-Fidelity PCR Master Mix (provides 1.5 mM MgCl2 in the final 1X concentration)
  • Water, nuclease-free
  • DMSO
Shelf Life24 Months
Storage buffer20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, stabilizers, 200 µg/mL BSA, and 50% glycerol.
Storage Condition-20 C
Gel image comparing Phusion Hot Start II DNA Polymerase to other suppliers' polymerases

Phusion Hot Start II DNA Polymerase provides extreme specificity and abundant yields.

Gel image comparing Phusion Hot Start II DNA Polymerase to other suppliers' polymerases

Five proofreading DNA polymerases from major suppliers were used to amplify 1.7 to 2.3 kb amplicons from human genomic DNA. All amplifications were performed in accordance with manufacturers instructions. Phusion Hot Start II DNA Polymerase provided high yields of specific products whereas all other enzymes delivered lower or no yields, some of them also amplifying non-specific products.

Superior yields of long products

Superior yields of long products

Superior yields of long products

20 kb fragment from λ DNA and 7.5 kb fragment from human genomic DNA was amplified with different hot start high fidelity PCR master Mixes. Phusion Green Hot Start II PCR Master Mix was the only master mix capable of providing high amounts of desired PCR products. In contrast, master mixes from other suppliers delivered zero or significantly lower yields and in some cases non-specific products.


Polymerase structure

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PCR applications

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  7. B. Dummitt et al., Yeast glutamine-fructose-6-phosphate aminotransferase (Gfa1) requires methionine aminopeptidase activity for proper function. J Biol Chem 280, 14356–14360 (2005).
  8. C. S. Fernandez et al., Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost. J Virol. 79, 5721–5731 (2005).
  9. S. Fiorucci et al., A FXR-SHP regulatory cascade modulates TIMP-1 and MMPs expression in HSCs and promotes resolution of liver fibrosis. J Pharmacol Exp Ther 314, 584-595 (2005).
  10. L. Fredriksson et al., Structural requirements for activation of latent platelet-derived growth factor-CC by tissue plasminogen activator. J Biol Chem. 280, 26856–26862 (2005).
  11. M. Gauster et al., Endothelial lipase is inactivated upon cleavage by the members of the proprotein convertase family. J Lipid Res 46, 977–987 (2005).
  12. R. A. Hoskins et al., Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP). Nucleic Acids Res. 33, e185 (2005).
  13. N. Ivashikina et al., AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive. J Gen Physiol. 125, 483–492 (2005).
  14. S. M. Julio, P. A. Cotter, Characterization of the filamentous hemagglutinin-like protein FhaS in Bordetella bronchiseptica. Infect Immun. 73, 4960-4971 (2005).
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  16. K. Moon et al., Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT. J Bacteriol. 187, 5732-5741 (2005).
  17. T. Nawy et al., Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell. 17, 1908–1925 (2005).
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  19. G. Rizzo et al., The methyl transferase PRMT1 functions as co-activator of farnesoid X receptor (FXR)/9-cis retinoid X receptor and regulates transcription of FXR responsive genes. Mol Pharmacol. 68, 551-558 (2005).
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454 sequencing

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