UA Biophysics:Protocols:Q5

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The Q5 High- Fidelity 2X Master Mix requires only the addition of primers and DNA template. It features a high-fidelity, thermostable DNA polymerase with 3´→ 5´ exonuclease activity. (Take GC% into account).

With an error rate > 100-fold lower than that of Taq DNA Polymerase, Q5 High-Fidelity DNA Polymerase is ideal for cloning and can be used for long or difficult amplicons. (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).

When used at the recommended 1X final concentration, the Q5 High-Fidelity Master Mix contains 2 mM MgCl2. (Take this concentration into account when designing primers. Since this is a master mix, you cannot decrease this concentration).

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
Q5 High-Fidelity 2X Master Mix 10 µL 1X
DMSO (optional*) 0.6 µL 3%
10 µM Forward Primer 1 µL 0.5 µM**
10 µM Reverse Primer 1 µL 0.5 µM**
Template DNA Variable*** < 1 µg
Nuclease-Free Water to 20 µL


* DMSO is a PCR enhancing agent used for difficult templates. It can be added up to a concentration of 10%.
** You can manipulate your primers' concentration according to your design.
*** 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 0.4 ng to 400 ng
Plasmid or Viral DNA 0.4 pg to 0.4 ng


(CAREFUL! You should add reagents in a completely different order to prevent contamination and primer degradation:

  1. Water
  2. F Primer
  3. R Primer
  4. Template DNA
  5. DMSO
  6. Q5 High-Fidelity 2X Master Mix)


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 20-30 seconds/kb
Final Extension 72 ºC 2 minutes
Hold 4 ºC -


* Denaturation time should be kept at minimum. 10 seconds usually work.

Please, do not hesitate to contact me if you have any question.

Manuela Vanegas-Ferro


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