UA Biophysics:Protocols:Phusion

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Phusion High-Fidelity DNA Polymerase offers both high-fidelity and robust performance, and thus is an ideal choice for cloning and can be used for long or difficult amplicons. (But take GC% into account).

With an error rate >50-fold lower than of Taq DNA Polymerase, Phusion is one of the most accurate thermostable polymerases available. Phusion DNA Polymerase possesses 5'→3' polymerase activity, 3'→5' exonuclease activity and will generate blunt-ended products. (Q5 has an error rate 2-fold lower than Phusion, but Phusion is slightly faster and does not come in a master mix. PCR Troubleshooting).

To determine the optimal annealing temperatures for a given set of primers, use of the NEB TmCalculator is highly recommended. (A must!)

Before setting up your reaction, take a look at this page. CAREFUL! The quantities of reagents and cycling conditions may be different, but their guidelines are important. University of Arizona

If you are having trouble with your PCR, you can visit Bitesize Bio. They have useful tips.

1. Reaction Setup: Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed and centrifuged prior to use.

Component 20 µL Reaction Final Concentration
Nuclease-free water to 20 µL
5X Phusion HF or GC Buffer 4 µL 1X
10 mM dNTPs 0.4 µL 200 µM
10 µM Forward Primer 1 µL 0.5 µM*
10 µM Reverse Primer 1 µL 0.5 µM*
Template DNA Variable**** < 1 µg
DMSO (optional**) 0.6 µL 3%
Phusion DNA Polymerase 0.2 µL*** 1 unit / 50 µL PCR

* You can manipulate your primers' concentration according to your design.
** DMSO is a PCR enhancing agent used for difficult templates. It can be added up to a concentration of 10%.
*** This volume can lead to pipetting error. Set up a master mix for all the reactions you need and then you can distribute it into the reaction tubes. This way you will pipette larger volumes and avoid some error.
**** Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:
DNA Amount
Genomic DNA 20 ng to 100 ng
Plasmid or Viral DNA 0.4 pg to 0.4 ng

(CAREFUL! You should add reagents in the order listed above to prevent contamination and primer degradation)

2. Gently mix the reaction and spin down in microcentrifuge. (Spinning is important in order to collect all the reagents in the bottom of the tube).

3. Cycling Conditions:

Step Temperature Time
Initial Denaturation 98 ºC 30 seconds
25-35 cycles 98 ºC 5-10 seconds
50-72 ºC (NEB TmCalc) 10-30 seconds*
72 ºC 15-30 seconds/kb**
Final Extension 72 ºC 2 minutes
Hold 4 ºC -

* Denaturation time should be kept at minimum. 10 seconds usually work.
** This polymerase is very fast. It will probably work with 15 seconds/kb.

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