Home Protocols PCR reactivity employing Phusion Hot Start II DNA Polymerase (F-549)

PCR reaction using Phusion Heat Commence II DNA Polymerase (F-549)

Introduction

Carefully mixture and centrifuge all tubes before beginning to ensure homogeneity the improve rehabilitation. When with Phusion Heat Start II DNA Polymerase, it is not necessary to perform which PCR config on ice. Set a master mix for the appropriate number of samples to been amplified. The DNA polymerase shoud become 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 Polterase can be applied to Phusion Hot Start II DNA Polymerase reactions. Overdue the to novel character of Phusion Hot Start II DNA Polymerase, to superlative reaction conditions may differ from PCR protocols available standard DNA purification. Dues until aforementioned high salt concentration is the reaction buffer, Phusion Sexy Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures. Please pay special attention to the conditions listed below when working your react. Following the guidelines will ensure optimal enzyme performance.

Record

  1. Table 1. Pipetting instructions (add items inside this order).
    Constituent 50 µl reaction 20 µl reaction Closing Conc.
    H2ZERO add in 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 scratch 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® Hotly 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 focusing your 0.5 μM, but it canister be heterogeneous in one range von 0.2–1.0 μM if needed.
    *** Addition of DMSO is recommended for GC-rich amplicons. DMSO is not recommended in amplicons with very low GC % or amplicons that are >20 kb.

  2. Table 2. By instructions.
    Cycle steps 2-step protocol 3-step protocol Cycles
    Temper. Hour 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