Thermo Scientific™ Phusion High-Fidelity PCR Master Mix with HF or GC Buffer

Catalog No.	Includes	No. of Reactions
F532L	GC Buffer	500 Reactions
F532S	GC Buffer	100 Reactions
F531L	HF Buffer	500 Reactions
F531S	HF Buffer	100 Reactions

Catalog No. F532L Supplier Thermo Scientific™ Supplier No. F532L

Description

Thermo Scientific Phusion High-Fidelity DNA polymerases set a gold standard for high performance PCR.

Thermo Scientific Phusion High-Fidelity DNA polymerases set a gold standard for high performance PCR. Featuring an error rate 50-fold lower than that of Taq and 6-fold lower than that of Pfu, Phusion High-Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity. Phusion DNA polymerases offer robust performance with short protocol times, even in the presence of PCR inhibitors, and generate higher yields with lower enzyme amounts than other DNA polymerase.

Phusion High-Fidelity PCR Master Mix is convenient 2X mix containing Phusion DNA Polymerase, nucleotides, and optimized reaction buffer including MgCl₂. Two master mix formulations are available: with HF Buffer (F-531S and F-531L) and with GC Buffer (F-532S and F-532L). The error rate of Phusion DNA Polymerase in HF Buffer (4.4 x 10^-7) is lower than that in GC Buffer (9.5 x 10^-7). Therefore, the master mix with HF Buffer should be used as the default master mix for high-fidelity amplification. However, GC Buffer can improve the performance of Phusion DNA Polymerase on some difficult or long templates, such as GC-rich templates or those with complex secondary structures.

Highlights

• High fidelity (25X Taq)
• Fast PCR due to short extension times (15–30 s/kb)
• Robust performance, minimal optimization needed

Applications

• Amplification of difficult (GC-rich) templates
• High-fidelity PCR
• Cloning
• Template generation for sequencing
• Long-range PCR (up to 20 kb)
• Mutagenesis
• High throughput PCR

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: • 2X Phusion Master Mix with GC Buffer (10 x 1.25 mL)
  • 100% DMSO (2 x 500 μL)
  
  Store at –20°C.
• GC-Rich PCR Performance: High
• Polymerase: Phusion High-Fidelity DNA Polymerase
• Reaction Speed: Fast
• Product Type: High-Fidelity PCR Master Mix
• Quantity: 500 reactions
• Shipping Condition: Dry Ice
• For Use With (Application): Standard PCR, High-fidelity PCR
• Fidelity (vs. Taq): 52X
• Hot Start: No
• No. of Reactions: 500 Reactions
• Overhang: Blunt
• Reaction Format: SuperMix or Master Mix
• Size (Final Product): 20 kb or less
• Includes: GC Buffer

Frequently Asked Questions (FAQs)

Do Phusion DNA Polymerases add the non-template dependent 3'-A overhang?

Phusion DNA Polymerases generate blunt end products; therefore, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. Taq DNA Polymerase (Cat. No. EP0401). However, before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.

Can Phusion DNA Polymerases extend at 1 second/kb?

Yes it is possible, especially when amplifying smaller amplicons. Processivity of Phusion DNA Polymerases is 10 times that of Pfu. We recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases, significantly shorter extension times (0-5 s/kb) can be used without compromising the yield. What separates Phusion Flash DNA Polymerase from other fast polymerases is that all steps in the PCR protocol can be shortened, including annealing and denaturation. This results in extremely fast protocols as compared with any other polymerase.

Can protocols optimized for Phusion DNA Polymerase be directly applied to Phusion Hot Start II DNA Polymerase?

Yes, protocols optimized for Phusion DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions.

Do you have any ready-to-use 2X Master Mix as an alternative to Phusion High-Fidelity PCR Kit (Cat. No. F553L)?

We offer Phusion High-Fidelity PCR Master Mix with HF Buffer (Cat. No. F531L) in a 2X Master Mix format. For GC-rich templates, the Phusion High-Fidelity PCR Master Mix with GC Buffer (Cat. No. F532L) can be used.

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