Phusion™ Hot Start II DNA Polymerase & dNTP Mix (10 mM each)

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).

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.

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.

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

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.