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Amplifying Control Templates using Phusion High-Fidelity PCR Kit

Protocol

  1. Reaction conditions:
    Pipetting instructions for control reactions
    Component Volume/
    50 µl reaction    		 Volume/
    20 µl reaction    		 Final Conc.
    H2O 34 µl 13.6 µl  
    5x Phusion HF Buffer 10 µl 4 µl 1X
    10 mM dNTPs 1 µl 0.4 µl 200 µM each
    Primers * 2.5 µl 1 µl 0.02 µM
    Control template DNA 2 µl 0.8 µl  
    Phusion DNA Polymerase 0.5 µl 0.2 µl ** 0.02 U/µl
    * Either the 1.3 kb primer set or 10 kb primer set.
    ** Dilution of polymerase should be made to 1x reaction bufer to avoid pipetting errors.
  2. Cycling conditions:
    A separate cycling protocol is given for both 1.3 kb and 10 kb control amplicons. Alternatively, both control reactions can be amplified simultaneously using the 10 kb cycling protocol. 

    Cycling conditions for 1.3 kb control fragment (2-step protocol)
    Cycle step Temp. Time Number of cycles
    Initial denaturation 98°C 1 min 1
    Denaturation
    Annealing/Extension    		 98°C
    72°C    		 5 s
    20 s    		 25
    Final extension
         		 72°C
    10°C    		 10 min
    hold    		 1

    Cycling conditions for 10 kb fragment (3-step protocol). This program can also be used if both control reactions are amplified simultaneously
    Cycle step Temp. Time Number of cycles
    Initial denaturation 98°C 1 min 1
    Denaturation
    Annealing
    Extension    		 98°C
    60°C
    72°C    		 5 s
    15 s
    2 min 30 s    		 15
    Final extension
         		 72°C
    10°C    		 10 min
    hold    		 1
    The cycling protocols above are recommendations. If you wish to run these controls together or with your experimental samples, please note that the controls have been shown to work in a variety of conditions. The 1.3 kb control has been successfully amplified with both 2- and 3-step protocols with extension times ranging from 15 s to 5 min, and cycle numbers ranging from 20 to 30. The 10 kb control has been successfully amplified with 3-step protocol with extension times ranging from 2 min to 5 min, and cycle numbers ranging from 20 to 30.
  3. Analysis of the control reactions:

    In the image on the left both control reactions have been run on an ethidium bromide stained agarose gel (1% SeaKem LE agarose in TAE buffer). For this run 15 μl of loading dye was added to the 50 μl control PCR reactions, and 5 μl of the resulting mixtures were loaded on the gel.
    Lane 1. DNA size standard
    Lane 2. 1.3 kb control amplicon
    Lane 3. 10 kb control amplicon
    Lane 4. DNA size standard

    After running your control reactions on a gel, compare the results to the image on the left to check for specificity and efficiency of the reactions.