Phusion Green Hot Start II High-Fidelity PCR Master Mix, 500 reactions - FAQs

View additional product information for Phusion Hot Start II High-Fidelity PCR Master Mixes - FAQs (F565S, F566S, F565L, F566L)

9 product FAQs found

What is the difference between Phusion Flash High-Fidelity PCR Master Mix and Phusion Hot Start II High-Fidelity PCR Master Mix?

Phusion Hot Start II High-Fidelity PCR Master Mix is a 2X ready-to-use solution based on Phusion Hot Start II DNA Polymerase. It is designed for the highest fidelity (52X Taq) and specificity. Phusion Flash High-Fidelity PCR Master Mix is based on a modified Phusion Hot Start II DNA Polymerase, which allows for extremely short cycling times and features somewhat lower fidelity (25X Taq).

Which buffer is used in Phusion Hot Start II High-Fidelity PCR Master Mix?

Phusion Hot Start II High-Fidelity PCR Master Mix is based on Phusion HF buffer. For amplification of GC-rich targets, we recommend to add 3% DMSO.

What is enzyme concentration in Phusion Hot Start II High-Fidelity PCR Master Mix?

Phusion Hot Start II DNA polymerase concentration is optimized to give good results in most reactions. When the PCR reaction is set up according to the instructions, the final concentration of Phusion enzyme is 1 U in 50 µL reaction (0.4 U in 20 µL reaction).

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 Phire and Phusion Hot Start II DNA Polymerases need a separate activation step in the PCR protocol?

No separate activation step is required since Phire and Phusion Hot Start II DNA Polymerases are inactivated by a reversibly bound, specific Affibody ligand that dissociates during initial denaturation.

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.