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Thermo Scientific Phire Hot Start II DNA Polymerase is an enhanced PCR enzyme for routine and high throughput PCR applications. It outperforms every Taq-based hot start polymerase. Phire Hot Start II DNA Polymerase is significantly faster, extremely robust, and also capable of amplifying long DNA fragments with high yields. These features are achieved through advanced protein engineering of the polymerase. It incorporates a unique double-stranded DNA binding domain which allows short extension times (10 to 15 s/kb), improves yields, and increases fidelity 2-fold compared to Taq DNA polymerase.
The hot start modification of Phire Hot Start II DNA Polymerase is based on the Affibody inactivation technology. This technology increases the specificity of the PCR reaction with no additional time required for initial activation of the enzyme.
Phire Green format is a combination of Phire Hot Start II DNA Polymerase and 5X Phire Green Buffer. The buffer includes a density reagent and two tracking dyes for direct loading of PCR products on a gel. The colored buffer does not interfere with enzyme performance and is compatible with downstream applications such as DNA sequencing, ligation and restriction digestion.
Phire Hot Start II PCR Master Mixes are convenient 2X mixes designed to minimize the number of pipetting steps. The master mixes contain Phire Hot Start II DNA Polymerase, nucleotides and optimized reaction buffer including MgCl2. Only template and primers need to be added to PCR reaction.
The optimal annealing temperature for Phire DNA Polymerases may differ significantly from that of Taq-based polymerases.
For optimal results start by accurately calculating your Tm with our Tm calculator.
Phire or Phire Green Hot Start II DNA Polymerase
Phire or Phire Green Hot Start II PCR Master Mix
A 1.5 kb fragment from the human cathepsin K gene was amplified with five different hot start DNA polymerases. Phire Hot Start II DNA Polymerase amplified high amounts of specific PCR product in just 29 minutes. In contrast, the PCR protocols for hot start Taq DNA polymerases from four major suppliers (A through D) were substantially longer and resulted in lower product yields.
A 2 kb fragment of human β-globin gene was amplified with different hot start PCR master mixes. Phire Green Hot Start II PCR Master Mix delivered high yields of specific product in just 41 minute whereas all other master mixes provided lower yields and in most cases required longer protocols.
Five genomic DNA fragments of different lengths were amplified with three different hot start DNA polymerases. Phire Hot Start II DNA Polymerase produced all five amplicons with very high yields. The competing hot start Taq DNA polymerases produced significantly lower yields and failed to amplify the 7.5 kb fragment.1 – 0.6 kb fragment of human glutathione peroxidase 3 gene2 – 1.0 kb fragment of human glutathione peroxidase 3 gene3 – 1.5 kb fragment of human cathepsin K gene4 – 2.7 kb fragment of human ß-2-microglobulin gene 5 – 7.5 kb fragment of human ß-globin gene
Five genomic DNA fragments of different lengths were amplified with different hot start PCR master mixes. Phire Green Hot Start II PCR Master Mix produced all five amplicons with high yields. Other hot start PCR master mixes produced lower or no yields, with some also amplifying non-specific products. 1 – 0.47 kb fragment of human calpain 10 gene 2 – 1.1 kb fragment of human KLK3 gene 3 – 1.7 kb fragment of human PPAR gamma gene 4 – 3.5 kb fragment of human plasminogen activator gene 5 – 7.5 kb fragment of human hemoglobin B gene
A 600 bp fragment from human genomic DNA was amplified with five different hot start DNA polymerases. With Phire Hot Start II DNA Polymerase, the PCR protocol was completed up to four times faster than with Taq DNA polymerases utilizing chemically modified or antibody-based hot start technologies (suppliers A through D). Green buffer further reduces experimental time by eliminating one pipetting step and allowing for direct loading on gel.
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