Thermo Fisher Scientific Lyo-ready Phusion Hot Start II High-Fidelity DNA Polymerase User guide

Brand: Thermo Fisher Scientific

Model: Lyo-ready Phusion Hot Start II High-Fidelity DNA Polymerase

Type: User guide

USER GUIDE Pub. No. MAN0017414 Rev. A.0

Thermo Scientiﬁc™ Lyo-ready Phusion™ Hot Start II High-Fidelity DNA Polymerase

Enzyme characteristics

Hot-start: Afbody™ technology

Length: Up to 20kb

Fidelity vs. Taq:>50X

Format: Separate components

PCR setup

Component 50-µL rxn Custom Final conc.

Water, nuclease-free to 50µL to µL —

5X Phusion™ Buffer110µL µL 1X

10mM dNTP mix 1 µL µL 0.2mM each

10µM forward primer22.5µL µL 0.5µM

10µM reverse primer2 2.5µL µL 0.5µM

Template DNA3varies varies varies

DMSO (optional)41.5µL µL 3%

Lyo-ready Phusion™ Hot StartII DNA

Polymerase (2U/µL) 0.5µL µL 0.02U/μL

1 5X Phusion™ HF Buffer is the default reaction buffer for high-delity amplication.

5XPhusion™ GC Buffer can be used for GC-rich templates or those with complex secondary

structures (see “Optimization strategies” for more information). 5XPhusion™ HF Buffer and

5X Phusion™ GC Buffer both contain 7.5 mM MgCl2

2 The recommendation for nal primer concentration is 0.5μM, but it can be varied in the

range of 0.2–1.0μM if needed.

3 50–250ng gDNA or 1pg–10ng plasmid DNA per 50-μL reaction (see “Optimization

strategies” for more information).

4 Addition of DMSO is recommended for GC-rich (>65%) amplicons. DMSO is not

recommended for amplicons with very low GC content or amplicons that are >20kb.

PCR protocol

See page 2 and page 3 to prepare and run your PCR experiment.

Optimization strategies

Click here for guidelines to optimize your PCR experiment.

Purchaser notiﬁcation

Click here for Limited Warranty, Disclaimer, and Licensing information.

Contents Sample Kit No.

EP1990SMP1

Size

500Units Kit contents

Storage

conditions ∤Store all contents at –20°C. Product is designed to withstand at

least 10 freeze-thaw cycles.

Required

materials

∤Template: gDNA, plasmid DNA, phage DNA, cDNA

∤Forward and reverse gene-specic primers

∤Invitrogen™ E-Gel™ General Purpose Gel, 1.2%

(Cat.No.G501801)

∤Invitrogen™ TrackIt™ 1kb Plus DNA Ladder (Cat.No.10488085)

∤0.2 or 0.5-mL nuclease-free microcentrifuge tubes

∤Gel loading buffer

∤Water, nuclease-free

Timing Varies depending on amplicon length.

Product

description

∤Thermo Scientic™ Lyo-ready Phusion™ Hot Start II DNA

Polymerase combines unique proofreading enzyme properties

and compatibility with lyophilization. Lyo-ready enzyme

formulation contains minimal amount of glycerol (≤0.5%) and

retains all favorable standard Phusion™ DNA Polymerase (with

50% glycerol) properties– high delity, increased processivity,

and resistance to common reaction inhibitors. The combination

of these features offers high exibility in terms of lyophilized

enzyme format that meets high performance requirements for

detection assays.

∤The enzyme combines DNA polymerase and a reversibly

bound, specic Afbody™ protein, which inhibits the DNA

polymerase activity at ambient temperatures, thus preventing

the amplication of non-specic products.

∤The Afbody™ ligand also inhibits the 3’to5’ exonuclease

activity of the polymerase, preventing degradation of primers

and template DNA during reaction setup. At polymerization

temperatures, the Afbody™ molecule is released, rendering

the polymerase fully active.

∤Phusion™ Hot StartII DNA Polymerase has 5'to3' polymerase

and 3'to5' exonuclease activities. It produces blunt-end DNA

products.

Important

guidelines Click here for important PCR guidelines.

Online

resources For further information, contact LCSVilnius@thermosher.com.

For Research Use Only. Not for use in diagnostic procedures.

Print Options

-2-

PCR procedure

The example PCR procedure below shows appropriate volumes for a single 50-µL reaction. For multiple reactions, prepare a master mix of components common to all reactions

to minimize pipetting error, then dispense appropriate volumes into each 0.2-mL or 0.5-mL PCR tube before adding template DNA and primers. When using the lyo-ready

Phusion™ Hot Start II DNA Polymerase, the PCR setup can be performed at room temperature.

Step Action Procedure details

1 Thaw reagents Thaw, mix, and briey centrifuge each component before use.

2 Prepare PCR mix

a. Add the following components to each PCR tube.

Note: Consider the volumes for all components listed in the table to determine the correct amount of water

required to reach your nal reaction volume.

Component 50-µL rxn Final conc.

Water, nuclease-free to 50µL —

5X Phusion™ HF Buffer110µL 1X

10mM dNTP mix 1µL 0.2mM each

10µM forward primer2 2.5µL 0.5μM

10µM reverse primer22.5µL 0.5μM

Template DNA3varies varies

DMSO (optional)41.5µL 3%

Lyo-ready Phusion™ Hot StartII DNA Polymerase 0.5µL 0.02U/μL

1 5X Phusion™ HF Buffer is the default reaction buffer for high-delity amplication. 5 X Phusion™ GC Buffer can be used

for GC-rich templates or those with complex secondary structures (see “Optimization strategies”, page 1). 5XPhusion

HF Buffer and 5X Phusion GC Buffer both contain 7.5 mM MgCl2.

2 The recommendation for nal primer concentration is 0.5μM, but it can be varied in the range of 0.2–1.0μM if needed.

3 50–250ng gDNA or 1pg–10ng plasmid DNA per 50-μL reaction (see “Optimization strategies”, page 1).

4 Addition of DMSO is recommended for GC-rich amplicons. DMSO is not recommended for amplicons with very low GC

content or amplicons that are >20kb.

b. Cap each tube, mix, then briey centrifuge the contents.

20 September 2018

For support, visit thermoﬁsher.com/support.

-3-

Step Action Procedure details

Incubate reactions in a

thermal cycler

Step

2-step protocol 3-step protocol

Temp. Time Temp.Time

Initial denaturation 98°C 30sec 98°C 30sec

25–35

PCR

cycles

Denature 98°C 5–10sec 98°C 5–10sec

Anneal1— — varies 10sec

Extend 72°C 15–30 sec/kb 72°C 15–30sec/kb

Final extension 72°C 5–10min 72°C 5–10min

4°C hold 4°C hold

1 IMPORTANT! The optimal annealing temperature for Phusion™ Hot Start II DNA Polymerase may differ signicantly from

that of Taq-based polymerases. Always use the Tm calculator at thermosher.com/tmcalculator to calculate the Tm of your

primers and the recommended annealing temperature. The 2-step protocol is recommended when primer Tm values are at

least 69°C (>20nt) or 72°C (≤20nt), when calculated with our Tm calculator.

Note: Refer to “Optimization strategies”, page 1, for guidelines to optimize cycling conditions.

Add gel loading buffer and

analyze with gel

electrophoresis

a. Add gel loading buffer to 10µL of PCR product, mix, then briey centrifuge the contents.

b. Analyze the sample using agarose gel electrophoresis.

c. Use your PCR product immediately in down-stream applications, or store it at –20°C.

Thermo Fisher Scientific Lyo-ready Phusion Hot Start II High-Fidelity DNA Polymerase User guide

Thermo Fisher Scientific Lyo-ready Phusion Hot Start II High-Fidelity DNA Polymerase User guide

Thermo Fisher Scientific Lyo-ready Phusion Hot Start II High-Fidelity DNA Polymerase User guide

Related papers

Other documents