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PCR reaction using Phusion Hot Start II DNA Polymerase (F-549)

Introduction

Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery. When using Phusion Hot Start II DNA Polymerase, it is not necessary to perform the PCR setup on ice. Prepare a master mix for the appropriate number of samples to be amplified. The DNA polymerase should be pipetted carefully and gently as the high glycerol content (50 %) in the storage buffer may otherwise lead to pipetting errors. 

Protocols optimized for Phusion® DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions. Due to the novel nature of Phusion Hot Start II DNA Polymerase, the optimal reaction conditions may differ from PCR protocols for standard DNA polymerases. Due to the high salt concentration in the reaction buffer, Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures. Please pay special attention to the conditions listed below when running your reactions. Following the guidelines will ensure optimal enzyme performance.

Protocol

  1. Table 1. Pipetting instructions (add items in this order).
    Component 50 µl reaction 20 µl reaction Final Conc.
    H2O add to 50 µl add to 20 µl  
    5x Phusion® HF Buffer* 10 μl 4 μl 1X
    10 mM dNTPs 1 μl 0.4 μl 200 μM each
    primer A** x µl x µl 0.5 µM
    primer B** x µl x µl 0.5 µM
    template DNA x µl x µl  
    (DMSO***, optional) (1.5 μl) (0.6 μl) (3 %)
    Phusion® Hot Start II
    DNA Polymerase (2 U/μl)    		 0.5 μl 0.2 μl 0.02 U/μl
    * 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 recommended for amplicons with very low GC % or amplicons that are >20 kb.

  2. Table 2. Cycling instructions.
    Cycle step 2-step protocol 3-step protocol Cycles
    Temp. Time Temp. Time
    Initial denaturation 98°C 30 s 98°C 30 s 1
    Denaturation
    Annealing
    Extension    		 98°C
    –
    72°C    		 5-10 s
    –
    15-30 s/kb    		 98°C
    X°C
    72°C    		 5-10 s
    10-30 s
    15-30 s/kb    		 25-35
    Final extension
         		 72°C
    4°C    		 5-10 min
    hold    		 72°C
    4°C    		 5-10 min
    hold    		 1