Q5 PCR NEB

Reaction Setup

REAGENT	VOLUME (µL)	FINAL CONC.
10 µM Forward Primer	2.5 µL	0.5 µM
10 µM Reverse Primer	2.5 µL	0.5 µM
Template DNA	Y (1ng)	1 ng
Q5 High-Fidelity 2X Master Mix	25 µL	1X
Nuclease-free Water	20- Y

Final Volume = 50µL

Notes:

• Q5 High-Fidelity 2X Master Mix has an error rate > 100-fold lower than that of Taq DNA Polymerase

• Q5 High-Fidelity 2X Master Mix can be stored at -20°C. If precipitate forms after thawing, resuspend before use

• Remember to first dilute primers in nuclease-free water if starting with a higher concentration

• Do not exceed 50 µL final volume

Thermocycling Conditions

STEP	TEMPERATURE (°C)	TIME
		<6kb	≥6kb
Initial Denaturation	98	30 sec	1-3 min
30 Cycles denature anneal extend	98 50-71 72	10 sec 20 sec 10 sec per kb	10 sec 50 sec 1 min per kb
Final Extension	72	2 min	2 min
Hold	4-22

To find annealing temperature, follow steps on the NEB Tm Calculator, [1] which should yield a temperature 3°C above the Tm of the lower primer

Modifications

If annealing temperature is above 72°C, use two-step PCR protocol below

STEP	TEMPERATURE (°C)	TIME
		<6kb	≥6kb
Initial Denaturation	98	30 sec	1-3 min
30 Cycles denature anneal & extend	98 ≥72	10 sec 10 sec per kb	10 sec 1 min per kb
Final Extension	72	2 min	2 min
Hold	4-22

Note that the length of each step should be thought of as a gradient, not a hard cutoff at 6kb.

For more information, visit the NEB Q5 High-Fidelity 2X Master Mix Protocol [2]