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Phire Hot Start II DNA Polymerase for Fast PCR |
Fast PCR is a PCR technique that has shortened cycling times compared to traditional PCR. Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix and Thermo Scientific Phire Hot Start II DNA Polymerases aid in fast PCR by incorporating more nucleotides per binding event compared to traditional polymerases. This high processivity results in fast extension rates and shorter PCR cycles.
Fast-cycling PCR enzyme formats
Phire Hot Start II DNA Polymerase |
Phusion Flash High-Fidelity PCR Master Mix |
|---|---|
| Stand-alone enzyme | |
Colorless Green* | Colorless |
| Master mix | |
Colorless Green* | N/A |
*Contains green buffer that includes density reagent and two tracking dyes for direct loading of PCR products on gels.
Phire Hot Start II DNA Polymerase for fast PCR
Phire Hot Start II DNA polymerase is affibody-mediated hot-start PCR enzyme with improved speed, yields, and amplicon length. It is fused with a dsDNA-binding domain that increases enzyme's proccessivity, enables short extension times (10–15 sec/kb), and helps improve yields compared to a standard hot-start Taq DNA polymerase.
Features of Phire Hot Start II DNA Polymerase
- Fast enzyme with quick hot start (no separate activation step required)
- High inhibitor tolerance
- Higher yields and amplicons length than with hot-start Taq DNA polymerase
- Direct gel loading of PCR products, with green reaction buffer formats
Fast-cycling PCR
Phire Hot Start II DNA Polymerase has an extension time of 10 sec/kb, requires no separate activation step, and allows direct loading of PCR products onto gels. Due to these features, PCR protocols with Phire Hot Start II DNA Polymerase is significantly faster than protocols with Taq DNA polymerases (Figure 1).
Figure 1. Short cycling times with Phire Hot Start II DNA Polymerase. 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 chemical or antibody-based hot-start technologies (suppliers A–D). Green buffer further reduces experimental time by allowing fewer pipetting steps and direct gel loading.
Green reaction buffer for direct gel loading
Phire Green Hot Start II DNA Polymerase and PCR Master Mixes are supplied with 5x Phire Green Reaction Buffer that includes a density reagent and two tracking dyes for direct loading of PCR products on gels (Figure 2).
Figure 2. Reaction mixtures containing Phire Green Reaction Buffer. (A) Before electrophoresis. (B) Blue and yellow tracking dyes after electrophoresis.
Exceptional yields in a short time
Phire Hot Start II DNA Polymerase incorporates a dsDNA-binding domain that allows short cycling times and improves amplicons yields. High yields can be achieved with both Phire Hot Start II stand-alone enzyme (Figure 3) and PCR master mix formats (Figure 4).
Figure 3. High target yields with Phire Hot Start II DNA Polymerase. A 1.5 kb fragment from the human cathepsin K gene was amplified with 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 were substantially longer and resulted in lower product yields.
Figure 4. Excellent target yields with Phire Hot Start II PCR Master Mix. 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 41 minutes, whereas other master mixes provided lower yields and, in most cases, required lengthier protocols.
Longer amplicons compared to hot start Taq DNA polymerase
Up to 7.5 kb of gDNA and 20 kb of phage DNA can be amplified with Phire Hot Start II stand-alone enzyme and PCR master mix formats. Phire Hot Start II DNA Polymerase can amplify targets longer than a standard hot-start Taq DNA polymerases.
Figure 5. Higher amplicons length with Phire Hot Start II DNA Polymerase. 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 high yields whereas the other hot-start Taq DNA polymerases produced significantly lower yields and failed to amplify the 7.5 kb fragment. 1: 0.6 kb, 2: 1.0 kb, 3: 1.5 kb, 4: 2.7 kb, 5: 7.5 kb
Figure 6. Higher amplicon length with Phire Hot Start II PCR Master Mix. Five genomic DNA fragments of different lengths were amplified with 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 nonspecific products. 1: 0.47 kb, 2: 1.1 kb, 3: 1.7 kb, 4: 3.5 kb, 5: 7.5 kb
Fast-cycling PCR
Phire Hot Start II DNA Polymerase has an extension time of 10 sec/kb, requires no separate activation step, and allows direct loading of PCR products onto gels. Due to these features, PCR protocols with Phire Hot Start II DNA Polymerase is significantly faster than protocols with Taq DNA polymerases (Figure 1).
Figure 1. Short cycling times with Phire Hot Start II DNA Polymerase. 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 chemical or antibody-based hot-start technologies (suppliers A–D). Green buffer further reduces experimental time by allowing fewer pipetting steps and direct gel loading.
Green reaction buffer for direct gel loading
Phire Green Hot Start II DNA Polymerase and PCR Master Mixes are supplied with 5x Phire Green Reaction Buffer that includes a density reagent and two tracking dyes for direct loading of PCR products on gels (Figure 2).
Figure 2. Reaction mixtures containing Phire Green Reaction Buffer. (A) Before electrophoresis. (B) Blue and yellow tracking dyes after electrophoresis.
Exceptional yields in a short time
Phire Hot Start II DNA Polymerase incorporates a dsDNA-binding domain that allows short cycling times and improves amplicons yields. High yields can be achieved with both Phire Hot Start II stand-alone enzyme (Figure 3) and PCR master mix formats (Figure 4).
Figure 3. High target yields with Phire Hot Start II DNA Polymerase. A 1.5 kb fragment from the human cathepsin K gene was amplified with 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 were substantially longer and resulted in lower product yields.
Figure 4. Excellent target yields with Phire Hot Start II PCR Master Mix. 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 41 minutes, whereas other master mixes provided lower yields and, in most cases, required lengthier protocols.
Longer amplicons compared to hot start Taq DNA polymerase
Up to 7.5 kb of gDNA and 20 kb of phage DNA can be amplified with Phire Hot Start II stand-alone enzyme and PCR master mix formats. Phire Hot Start II DNA Polymerase can amplify targets longer than a standard hot-start Taq DNA polymerases.
Figure 5. Higher amplicons length with Phire Hot Start II DNA Polymerase. 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 high yields whereas the other hot-start Taq DNA polymerases produced significantly lower yields and failed to amplify the 7.5 kb fragment. 1: 0.6 kb, 2: 1.0 kb, 3: 1.5 kb, 4: 2.7 kb, 5: 7.5 kb
Figure 6. Higher amplicon length with Phire Hot Start II PCR Master Mix. Five genomic DNA fragments of different lengths were amplified with 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 nonspecific products. 1: 0.47 kb, 2: 1.1 kb, 3: 1.7 kb, 4: 3.5 kb, 5: 7.5 kb
Additional enzymes for fast PCR
Phusion Plus DNA polymerase is a high-fidelity DNA polymerase with universal annealing feature. This innovative feature allows co-cycling multiple DNA targets within the same protocol.
Phire Hot Start II DNA Polymerase manuals
Additional resources
Learning resources
Molecular biology workflow solutions
PCR web tools
Custom PCR enzymes
Stylesheet for Classic Wide Template adjustments
For Research Use Only. Not for use in diagnostic procedures.