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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.
Mrs. Evy Vanderheyden, working at the VIB/KULeuven Molecular Microbiology and Biotechnology department shares her positive experience with Q5 High-Fidelity DNA Polymerase for the robust amplification of genomic yeast DNA:
“Most important with doing PCR for us is a specific PCR product and a high yield. We need a proofreading enzyme since we sequence the obtained PCR products afterwards to check the correct sequences. We do not want errors introduced by the polymerase resulting in false positives or negatives.
For these PCR reactions we used ethanol precipitated genomic yeast DNA. We did a reaction with Phusion High- Fidelity DNA Polymerase like we used to do and compared this with the new Q5 polymerases with the same reaction conditions. It is clear to see that the yield obtained with Q5 High-Fidelity DNA Polymerase (both normal and Hot Start) is much better than the yield obtained with Phusion. After sequencing it turned out to be perfectly PCRed.”
Component | 25 µl Reaction | Final Concentration |
---|---|---|
5x Q5 Reaction Buffer |
5 µl |
1x |
10 mM dNTP per nucleotide |
0.5 µl each (2 µl total) |
800 µM each (200 µM total) |
10 μM Forward primer |
1.25 µl |
0.5 µM |
10 µM Reverse primer |
1.25 µl |
0.5 µM |
Template DNA |
1 µl |
70 ng |
Q5 High-Fidelity DNA Polymerase |
0.25 µl |
0.02 U/µl |
Nuclease-Free Water |
14.25 µl |
|
Step | Temperature | Time |
---|---|---|
Pre-denaturation |
98 °C |
30 seconds |
30 Cycles * |
98 °C |
15 seconds |
58 °C |
30 seconds |
|
72 °C |
30 seconds |
|
Final extension | 72 °C | 120 seconds |
Hold |
10 °C |
∞ |
Cat. # | Description | Quantity |
---|---|---|
100/500/2500 units |
||
100/500 rxns |
||
100/500/2500 units |
||
100/500 rxns |
||
50/250 rxns |
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6 ml |