Home Protocols PCR reaction using Phusion Blood Direct PCR Kit

PCR reaction using Phusion Blood Direct PCR Kit

Overview

Carefully mix and spin down all tubes before opening to ensure homogeneity and improve recovery. The PCR setup can be performed at room temperature.

Protocol

  1. Table 1. Pipetting instructions.
    Component 20 µl reaction 50 µl reaction Final Conc.
    H2O add to 20 µl add to 50 µl  
    2x Phusion® Blood PCR Buffer 10 µl 25µl 1X
    Primer A x µl x µl 0.5 µM
    Primer B x µl x µl 0.5 µM
    Phusion® Blood
    DNA Polymerase    		 0.4 µl 1 μl  
    Whole blood 1 μl 2.5 μl (see1.1)
    Optional components for reaction optimization
    50 mM MgCl2 0.6 μl 1.5 μl (see1.3)
    50 mM EDTA 0.5-1.0 μl 1.25-2.5 μl (see1.3)
    DMSO 1.0 μl 2.5 μl 5 % (see1.4)
  2. Table 2. Recommended cycling protocol.
    Cycle step  2-step protocol  3-step protocol Cycles
    Temp. Time Temp. Time
    Lysis of cells (see2.1) 98°C 5 min 98°C 5 min 1
    Denaturation
    Annealing (see 2.2)
    Extension(see2.3)    		 98°C
    --
    72°C    		 1 s
    --
    15-30 s/kb    		 98°C
    X°C
    72°C    		 1 s
    5 s
    15-30 s/kb    		 35-40
    Final extension
         		 72°C
    4°C    		 1 min
    hold    		 72°C
    4°C    		 1 min
    hold    		 1