PCR with Q5® Polymerase (M0491)

Abstract

This protocol describes methods for PCR using Q5® High-Fidelity DNA Polymerase (M0491).

Before start

Please note that protocols with Q5® High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.

Steps

Set up the following reaction On ice:

Note

All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.

A	B	C	D
COMPONENT	25 µl REACTION	50 µl REACTION	FINAL CONCENTRATION
5X Q5 Reaction Buffer	5 µl	10 µl	1X
10 mM dNTPs	0.5 µl	1 µl	200 µM
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
Template DNA	variable	variable	< 1,000 ng
Q5 High-Fidelity DNA Polymerase	0.25 µl	0.5 µl	0.02 U/µl
5X Q5 High GC Enhancer (optional)	(5 µl)	(10 µl)	(1X)
Nuclease-Free Water	to 25 µl	to 50 µl

Gently mix the reaction.

Collect all liquid to the bottom of the tube by a quick spin if necessary and overlay the sample with mineral oil if using a PCR machine without a heated lid.

Quickly transfer PCR tubes to a PCR machine preheated to the denaturation temperature (98°C) and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

A	B	C
STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
25–35 Cycles	98°C	5–10 seconds
	*50–72°C	10–30 seconds
	72°C	20–30 seconds/kb
Final Extension	72°C	2 minutes
Hold	4–10°C

*Use of the NEB Tm Calculator is highly recommended.

Abstract

Before start

Steps

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