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Key Concepts

PCR is one of the most important techniques used in molecular biology. It is used to copy a piece of DNA with a specific sequence and amplify it to obtain billions of copies of this sequence. In this section, we will use PCR to amplify the DNA sequence that codes for the Green Fluorescent Protein (GFP) from genomic DNA (gDNA) extracted from the jellyfish Aequorea victoria.


  • PCR tubes
  • 10X Phusion HF Reaction Buffer
  • dNTPs
  • Gibson primers (forward and reverse)
  • Aequorea victoria gDNA
  • Nuclease-free water
  • Phusion polymerase

*Phusion Buffer, dNTPs, primers, gDNA, and the Phusion polymerase are stored in the -20 °C freezer.


PCR Reaction Mixture


Stock concentration

Final reaction concentration

Amount to use (uL)

Phusion HF Reaction Buffer

10X 1X a


2.5 mM 0.2 mM


Gibson Forward Primer

10 µM 0.4 µM


Gibson Reverse Primer

10 µM 0.4 µM


Aequorea victoria gDNA

5 ng/µL 5 ng


Nuclease-free water


Phusion Polymerase

5 units/µL 10 units


Total Reaction Volume    

50 µL


Important note:  Enzyme stocks (e.g. polymerases) are extremely temperature-sensitive!  They should be kept on ice at ALL times when outside of the freezer.  Also, the enzyme is suspended in a high-percentage glycerol stock, which tends to stick to the sides of your tip. To minimize errors in pipetting these glycerol stocks, wipe the side of the tip off within the enzyme tube and only dip the pipette tip just below the surface.

  1. Add the above reagents in the order listed above to a PCR tube (The instructors will have the Phusion Polymerase at the front of the class).
  2. Mix the tube contents thoroughly by pipetting up and down a few times, pipetting gently to minimize the introduction of bubbles.
  3. Insert tube into thermal cycler and close the lid.
  4. Run the thermal cycler with the following program:

PCR Cycling Parameters


Temperature Time

Number of Cycles

a. Initial Denaturation


120 seconds

1 cycle

b. Denaturation


30 seconds

35 cycles 

(repeat steps b-d 

35 times)

c. Annealing


30 seconds

d. Extension


60 seconds

e. Hold



1 cycle