Thermo Fisher Scientific Phusion User Manual
USER GUIDE
Phusion™ Plus Green PCR Master Mix
Catalog Numbers F632S, F632L, F632XL
Pub. No. MAN0025048 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available from thermofisher.com/support.
Product description
The Thermo Scientific™ Phusion™ Plus Green PCR Master Mix is a ready-to-use mixture of DNA polymerase, salts, magnesium, and
dNTPs for ecient PCR amplification. The master mix is ideal for applications where accuracy is important (cloning, sequencing, site
directed mutagenesis).
Two tracking dyes and a density reagent are pre-added to the master mix for direct loading of PCR products onto gels.
The master mix contains the Phusion™ Plus DNA Polymerase, a proofreading DNA polymerase that combines a novel Pyrococcus-like
enzyme with a processivity-enhancing domain and universal primer annealing feature.
The polymerase is inactive at ambient temperatures due to an Abody™ molecule mediated hot start mechanism, allowing reaction setup
and storage of pre-assembled PCR reactions at room temperature. Enzyme activity is restored after the initial denaturation step.
Primer annealing is performed at 60°C because proprietary additives in the reaction buer stabilize primer-template duplexes during
annealing, and eliminate the need to optimize annealing temperature for each primer pair.
The polymerase possesses the following characteristics:
• 5´→3´ DNA polymerase activity.
• 3´→5´ exonuclease activity.
• Generates blunt end amplification products.
• Amplifies up to 10 kb from genomic DNA, and 20 kb from low complexity DNA.
• Works with both AT and GC rich targets (Phusion™ GC Enhancer is provided for amplicons with >65% GC content).
• >100X fidelity compared to Taq polymerase.
Contents and storage
Component
Phusion™ Plus Green PCR Master Mix 2 × 1.25 mL 10 × 1.25 mL 40 × 1.25 mL
Water 2 × 1.25 mL 10 × 1.25 mL 40 × 1.25 mL
F632S
100 reactions
F632L
500 reactions
F632XL
4 × 500 reactions
Storage
–25°C to –15°CPhusion™ GC Enhancer 1.25 mL 4 × 1.25 mL 16 × 1.25 mL
General guidelines
• Use 98°C for denaturation.
• Use 15–30 s/kb for extension.
• The polymerase cannot read through dUTP-derivatives or dITP in the template strand, thus primers and dNTP mixes containing such
nucleotides are not compatible.
• Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery. Prepare a master mix for the
appropriate number of samples to be amplified.
• Take precautions to avoid cross-contamination by using aerosol-resistant barrier tips and analyzing PCR products in a separate area
from PCR assembly.
For Research Use Only. Not for use in diagnostic procedures.
Required materials not supplied
• Template: genomic DNA, plasmid, phage DNA, cDNA
• Forward and reverse primers
• TopVision Agarose Tablets (Cat no. R2801)
• GeneRuler 1 kb DNA Ladder (Cat.no. SM0311)
• 0.2 or 0.5-mL nuclease-free microcentrifuge tubes
• Water, nuclease-free
Perform PCR
1. Prepare reaction by adding the following components in the order listed in the following table.
Component
2X Phusion™ Plus Green PCR Master Mix
[1]
20 µL rxn 50 µL rxn Final conc.
10 µL 25 µL 1X
Forward primer x µL x µL 0.5 µM
Reverse primer x µL x µL 0.5 µM
Template DNA x µL x µL
5X Phusion™ GC Enhancer
[3]
4 µL 10 µL 1X
Water, nuclease free add to 20 µL add to 50 µL —
[1]
Provides 1.7 mM MgCl2 at 1X concentration.
[2]
Reduce the primer concentration to 0.2 µM final concentration when amplifying >5 kb targets from genomic DNA and for multiplex reactions.
[3]
(Optional) recommended only for targets with >65% GC content.
2. Run a thermal cycler program set to the following parameters according to the protocol to be performed.
a. 3-step protocol
Cycle step
Temp. Time Cycles
Initial Denaturation 98°C 30 s 1
Denaturation
Annealing
Extension
98°C
60°C
72°C
5–10 s
10 s
15–30 s/kb
[2]
[2]
0.01–10 ng plasmid
5–100 ng genomic DNA
25–35
Final extension
72°C
4°C
5 min
Hold
b. 2-step protocol (for primers >30 nt in length)
Cycle step
Temp. Time Cycles
Initial Denaturation 98°C 30 s 1
Denaturation
Annealing/extension
Final extension
98°C
72°C
72°C
4°C
5–10 s
15–30 s/kb
5 min
Hold
2 Phusion
1
Hold
25–35
1
Hold
™
Plus Green PCR Master Mix User Guide
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