Protocol for a Routine PCR with Phusion® High-Fidelity PCR Kit

Introduction

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification. PCR amplifies specific DNA sequences exponentially by using multiple cycles of a three-step process. First, the double-stranded DNA template is denatured at a high temperature. Sequence-specific primers are then annealed to sites flanking the target sequence. A thermostable DNA polymerase extends the annealed primers, thereby doubling the amount of the original DNA sequence. This newly synthesized product then becomes an additional template for subsequent cycles of amplification. These three steps are repeated for 20 to 30 cycles, resulting in a 10⁵-10⁹ fold increase in target DNA concentration.

The unique Phusion High-Fidelity DNA Polymerase offers robust performance and can be used for all PCR applications. Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, increases fidelity and speed. Phusion DNA Polymerase is an ideal choice for cloning and can be used for long or difficult amplicons. With an error rate >50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase, Phusion is a highly accurate thermostable polymerase. Phusion DNA Polymerase possesses 5´→ 3´ polymerase activity, 3´→ 5´ exonuclease activity and will generate blunt-ended products.

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed and centrifuged prior to use. It is important to add Phusion DNA Polymerase last, in order to prevent any primer degradation caused by the 3´→5´ exonuclease activity. Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors. Please note that protocols with Phusion DNA Polymerase may differ from protocols with other standard polymerases. As such, conditions recommended below should be used for optimal performance.

Protocol

1. Set up the appropriate reactions on ice:

   COMPONENT	25 µl REACTION	50 µl REACTION	FINAL CONCENTRATION
   Phusion DNA Polymerase	0.25 µl	0.5 µl	1.0 units
   5X Phusion HF or GC Buffer	5 µl	10 µl	1X
   10 µM Forward Primer	1.25 µl	2.5 µl	0.5 µM
   10 µM Reverse Primer	1.25 µl	2.5 µl	0.5 µM
   10 mM dNTPs	0.5 µl	1 µl	200 µM
   Template DNA	variable	variable	< 250 ng
   DMSO (optional)	(0.75 µl)	(1.5 µl)	3%
   Nuclease-free water	to 25 µl	to 50 µl

   
   
   
2. Gently mix the reaction and spin down in microcentrifuge.
   If the thermocycler does not have a heated lid, overlay the sample with mineral oil.
   
   
   
3. Cycling Conditions for a Routine PCR:

   CYCLE STEP	CYCLES	TEMP	TIME
   Initial denaturation	1	98°C	30 seconds
   Denaturation Annealing Extension	30	98°C 45–72°C 72°C	5-10 seconds 10-30 seconds 15-30 seconds/kb
   Final extension	1	72°C	5–10 minutes
   Hold	1	4°C	∞