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Thermo Scientific™ Phusion U Multiplex PCR Master Mixes

Catalog No. F564S
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Catalog No. Color No. of Reactions
F564S Green 100 Reactions
F562S Colorless 100 Reactions
F564L Green 500 Reactions
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Catalog No. F564S Supplier Thermo Scientific™ Supplier No. F564S
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Description

Thermo Scientific Phusion U Multiplex PCR Master Mix is a ready-to-use, 2X end-point PCR master mix designed for simultaneous amplification of multiple targets in a single tube.

Thermo Scientific Phusion U Green Multiplex PCR Master Mix is a ready-to-use, 2X end-point PCR master mix designed for simultaneous amplification of multiple targets in a single tube. Over 20 primer pairs may be combined into a single reaction for highly specific and efficient multiplexing over a broad range of primer and template concentrations.

The Phusion U Multiplex PCR Master Mix contains a proprietary buffer with balanced concentrations of all PCR components eliminating the need for tedious optimization. The Green format further simplifies the workflow - it includes a density reagent and two electrophoresis tracking dyes for direct loading of PCR products on gels.

Highlights

  • Specific and efficient multiplex PCR of over 20 targets up to 2.5 kb
  • Excellent results with templates of suboptimal purity due to inhibitor tolerance
  • Fast cycling and direct loading of PCR products on gels

Applications

  • Genotyping
  • Pathogen detection
  • Food testing
  • Analysis of genetically modified organisms
  • Amplification of microsatellites

The master mix is based on Phusion U Hot Start DNA Polymerase, a high performance enzyme developed using fusion technology. Similarly to other enzymes from Phusion family, Phusion U DNA Polymerase incorporates more nucleotides per binding event than any other DNA polymerase. This allows achieving high yields of PCR products with shorter extention times and consequently reduced total protocol times. Significantly enhanced processivity also enables successful multiplex PCR on difficult targets such as GC-rich templates or templates of suboptimal purity. Being highly tolerant of many PCR inhibitors, Phusion U Multiplex PCR Master Mixes can even be used with unpurified samples such as blood or serum.

Specificity of multiplex PCR is increased by Affibody-mediated hot start mechanism. Reversibly bound Affibody ligand inhibits the polymerase at ambient temperatures preventing amplification of nonspecific products and formation of primer-dimers.

Using Phusion DNA Polymerases

Annealing rules for Phusion DNA Polymerases are different from many common DNA polymerases (such as Taq DNA polymerases).

Specifications

Concentration 2X
Content And Storage • Phusion U Hot Start DNA Polymerase
• 2X reaction buffer
• 3 mM MgCl2
• 400 μM of each dATP, dTTP, dCTP and dGTP

Master mix is supplied with nuclease-free water.
Store at -20°C.
GC-Rich PCR Performance High
Polymerase Phusion U Hot Start DNA Polymerase
Reaction Speed Fast
Product Type Multiplex PCR Master Mix
Quantity 100 x 50 μL Reactions
Shipping Condition Dry Ice
For Use With (Application) Hot-start PCR
Fidelity (vs. Taq) 25X
Hot Start Built-In Hot Start
No. of Reactions 100 Reactions
Overhang Blunt
Reaction Format SuperMix or Master Mix
Size (Final Product) 2.5 kb or less
Color Green
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Frequently Asked Questions (FAQs)

How much blood can be added directly to multiplex PCR reaction using Phusion U Multiplex PCR Master Mix?

For direct multiplex PCR without prior DNA purification, add up to 4 µL of whole blood to 50 µL final reaction volume.
Can Phusion U Multiplex PCR Master Mix be stored at +4 degrees C?

Yes, Phusion U Multiplex PCR Master Mix can be stored at +4 degrees C for up to 3 months with no effect on performance.
What is the limit of amplicon size using Phusion U Multiplex PCR Master Mix?

Phusion U Multiplex PCR Master Mix is designed for simultaneous amplification of targets up to 2.5 kb.
How many fragments can be amplified in a single reaction using Phusion U Multiplex PCR Master Mix?

We have demonstrated successful simultaneous amplification of 24 DNA fragments.
What are the Phusion U Multiplex PCR Master Mixes and what is the purpose of the Green format?

Thermo Scientific Phusion U Multiplex PCR Master Mixes (Cat. No. F562S, F564S, F562L, F564L) are ready-to-use, 2X end-point PCR master mixes designed for simultaneous amplification of multiple targets in a single tube. Over 20 primer pairs may be combined into a single reaction for highly specific and efficient multiplexing over a broad range of primer and template concentrations. Phusion U Multiplex PCR Master Mixes contains a proprietary buffer with balanced concentrations of all PCR components eliminating the need for tedious optimization.

The Green format of the Phusion U Multiplex PCR Master Mixes (Cat. No. F564S, F564L) further simplifies the workflow: it includes a density reagent and two electrophoresis tracking dyes for direct loading of PCR products on gels.
Can I use Phusion Green and Phire Green PCR products for subsequent applications such as sequencing, ligation, and restriction digestion?

Yes. The dyes do not interfere with downstream applications such as DNA sequencing, ligation, and restriction digestion.
What dyes are present in Phusion Green and Phire Green buffers?

Phusion Green and Phire Green buffers contain two dyes for monitoring electrophoresis progress. The blue dye migrates with 3-5 kb DNA fragments and the yellow dye migrates faster than 10 bp DNA fragments in 1% agarose gel. The dyes have excitation peaks at 424 nm and 615 nm, respectively.

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