Difference between revisions of "Q5 PCR NEB"

Latest revision as of 21:05, 14 June 2016

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

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

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]

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+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 [https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492]