Q5 High Fidelity DNA Polymerase

 

Q5 High-Fidelity DNA Polymerase sets a new standard for both high fidelity and robust performance. The Q5 buffer system is designed to provide superior performance with minimal optimization across a broad range of amplicons regardless of GC content.

Marjorie Mercier, a scientist in Brussels, shares her positive experience with Q5 High-Fidelity DNA Polymerase for the amplification of the IgM variable region.

We needed a polymerase that could handle templates with a varying GC content including GC rich. Microdessected germinal center cells were used to clone the V(D)J combination in IgM for expression in cells. Therefore we need the correct sequence to keep the ORF intact. We don’t want to introduce errors by the polymerase.

We performed a nested PCR on IgM transcripts yielding a PCR product from about 500-600 bp with variable GC content. We tested the new Q5 High Fidelity polymerase against Phusion High Fidelity polymerase. The results clearly show a much better yield for Q5 then Phusion.

The fragments have been completely sequenced and were devoid of any mistake.

 

Reaction Set-up

Component	25 µl Reaction	Final Concentration
5x Q5 Reaction Buffer	10 µl	1x
5 mM dNTP	1 µl each	200 µM each
10 µM Forward primer (1st or 2nd)	1 µl	0.5 µM
10 µM Reverse primer (1st or 2nd)	1 µl	0.5 µM
Template DNA (cDNA or 1st PCR product 1/100)	2.5 µl	< 50 ng
Q5 High-Fidelity DNA Polymerase	0.25 µl	0.5 U
Nuclease-Free Water	To 25 µl	To 25 µl

 

PCR Protocol

Step	Temperature	Time
Initial Denaturation	98 °C	30 seconds
20 / 25 Cycles	98 °C	10 seconds
	65 °C / 70 °C	30 seconds
	72 °C	15 seconds (+1 sec / cycle)
Final extension	72 °C	5 minutes
Hold	10 °C	0.5 U

Ordering Information

Cat. #	Description	Quantity
M0491S/L/XL	Q5™ High-Fidelity DNA Polymerase	100/500/2500 units
M0492S/L	Q5™ High-Fidelity 2X Master Mix	100/500 rxns
M0493S/L/XL	Q5™ Hot Start High-Fidelity DNA Polymerase	100/500/2500 units
M0494S/L	Q5™ Hot Start High-Fidelity 2X Master Mix	100/500 rxns
E0555S/L	Q5™ High-Fidelity PCR Kit	50/250 rxns
B9027S	Q5™ Reaction Buffer Pack	6 ml