Guidelines for Phusion DNA Polymerase High-Fidelity PCR Kit
Phusion DNA Polymerase (2U/μl) is provided with 5x Phusion HF Buffer and 5x Phusion GC Buffer. Both buffers contain 1.5 mM MgCl2 at final reaction concentrations. Separate tubes of DMSO and 50 mM MgCl2 solutions are provided for further optimization.
1. Basic reaction conditions for DNA amplification:
Carefully mix and centrifuge all tubes before opening to improve recovery. PCR reactions should be set up on ice. Phusion DNA Polymerase should be pipetted carefully and gently as the high glycerol content (50%) in the storage buffer may be otherwise lead to pipetting errors. It is critical that the Phusion DNA Polymerase is the last component added to the PCR mixture, since the enzyme exhibits 3´→5´ exonuclease activity that can degrade primers in the absence of dNTPs.
* Optionally 5x Phusion GC Buffer can be used, see section 4.2 for details.
** The recommendation for final primer concentration is 0.5 μM, but it can be varied in a range of 0.2-1.0 μM, if needed.
*** Addition of DMSO is recommended for GC-rich amplicons. DMSO is not ecommended for amplicons with very low GC % or amplicons that are >20 kb.
2. Cycling conditions:
Due to the novel nature of Phusion DNA Polymerase, optimal reaction conditions may differ from standard enzyme protocols. Phusion DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Following the guidelines will ensure optimal enzyme performance.