May 15, 2020
Version 1
PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515): General PCR, USER®Cloning, dUTP incorporation/Carryover prevention V.1
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/2019/07/02/pcr-using-q5u-hot-start-high-fidelity-dna-polymerase-neb-m0515
Protocol Citation: New England Biolabs 2020. PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515): General PCR, USER®Cloning, dUTP incorporation/Carryover prevention. protocols.io https://dx.doi.org/10.17504/protocols.io.7schnaw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 28, 2019
Last Modified: March 29, 2021
Protocol Integer ID: 28196
Keywords: Q5U, USER cloning, dUTP incorporation
Abstract
Q5U Hot Start High-Fidelity DNA Polymerase is a modified version of Q5®High-Fidelity DNA Polymerase, a novel thermostable DNA polymerase that possesses 3′ to 5′ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases.
Guidelines
General Guidelines:
1. Template:
Use of high quality, purified DNA templates greatly enhances the success of PCR.
Recommended amounts of DNA template for a 50 µl reaction are as follows:
| DNA | AMOUNT | |
| DNA Genomic | 1 ng – 1 µg | |
| Plasmid or Viral | 1 pg – 1 ng |
2. Primers:
Oligonucleotide primers are generally 20 – 40 nucleotides in length and ideally have a GC content of 40 – 60 %. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
3. USER DNA Engineering
Target DNA molecules and cloning vector are generated by PCR with 8 – 12 bases of homology between two fragments. PCR primers start with a 5′ A and contain a single deoxyuracil residue (dU) flanking the 3′ end of the homology region, and can be designed to accommodate multiple-fragment assembly, nucleotide substitutions, insertions and/or deletions. We recommend using the GeneDesign (http://genedesign.thruhere.net/gd/) software to design primers for USER junctions. The best results are typically seen when using each primer at a final concentration of 0.5 µM.
4. Mg++and additives:
Typically, the Mg++concentration for Q5U Hot Start High-Fidelity DNA Polymerase should be 2.0 mM. When used at a final concentration of 1X, the Q5U Reaction Buffer provides this optimal Mg++concentration. The addition of common PCR additives such as DMSO may improve amplification of certain difficult or long targets. In these cases, we recommend the addition of up to 2 % DMSO.
5. Deoxynucleotides:
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.
6. dUTP Incorporation/Carryover Prevention
Q5U Hot Start High-Fidelity DNA Polymerse is a dUTP-tolerant DNA polymerase that efficiently incorporates dUTP and amplifies uracil-containing substrates. To prevent carryover contamination, dUTP and Antartic Thermolabile UDG (NEB #M0372) can be added to the reaction. dTTP can be fully replaced by dUTP in the amplification of certain targets. For best results, we recommend adding dUTP at a final concentration of 200 μM. For UDG activation, a 10 minute, 25 °C incubation step should be added before the initial denaturation step. Typical cycling parameters can be used thereafter.
7. Q5U Hot Start High-Fidelity DNA Polymerase concentration:
We generally recommend using Q5U Hot Start High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5U Hot Start High-Fidelity DNA Polymerase may vary from 10 – 40 units/ml (0.5 – 2.0 units/50 μl reaction) depending on amplicon length and difficulty. It is rarely helpful to exceed 2.0 units/50 μl reaction, especially for amplicons longer than 5 kb.
8. Buffers:
The 5X Q5U Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. The 5X Q5U Reaction Buffer contains 2.0 mM Mg++at a final (1X) concentration.
9. Denaturation:
Q5U Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.
An initial denaturation of 30 seconds at 98 °C is sufficient for most targets being amplified from pure DNA templates. Longer initial denaturation times can be used (up to 3 minutes) for templates that require it. During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5 – 10 second denaturation at 98 °C is recommended for most templates.
10. Annealing:
Optimal annealing temperatures for Q5U Hot Start High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
For high Tm primer pairs, two-step cycling without a separate annealing step can be used (see note 11).
11. Extension:
The recommended extension temperature is 72 °C. Extension times are generally 20 – 30 seconds per kb for complex, genomic samples. Extension time can be increased to 1 minute per kb for long, complex templates, if necessary.
A final extension of 5 minutes at 72 °C is recommended.
12. Cycle number:
Generally, 30–35 cycles yield sufficient product. For genomic amplicons, 30 cycles are recommended.
13. 2-step PCR:
When primers with annealing temperatures ≥ 72 °C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
14. Amplification of long products:
When amplifying products > 6 kb, it is often helpful to increase the extension time to 1 minute /kb.
15. PCR product:
The PCR products generated using Q5U Hot Start High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5U Hot Start High-Fidelity DNA Polymerase will degrade any overhangs generated.
Materials
MATERIALS
Q5U® Hot Start High-Fidelity DNA PolymeraseNew England BiolabsCatalog #M0515
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Please note that protocols with Q5U Hot Start High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
Reaction Setup:
Q5U Hot Start High-Fidelity DNA Polymerase is inhibited at room temperature, allowing flexible reaction setup (room temperature or ice).
All components should be mixed prior to use.
General PCR, USER®Cloning, dUTP incorporation/Carryover prevention
General PCR, USER®Cloning, dUTP incorporation/Carryover prevention
Set up the reaction using the following table:
| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration | |
| 5X Q5U 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 | |
| Q5U Hot Start High-Fidelity DNA Polymerase | 0.25 µl | 0.5 µl | 0.02 U/µl | |
| 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. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Note
Q5U Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.
| STEP | TEMP | TIME | |
| Initial Denaturation | 98 °C | 30 seconds | |
| 30 Cycles | 98 °C | 5 – 10 seconds | |
| *55 – 72 °C | 20 seconds | ||
| 72 °C | 20 – 30 seconds/kb | ||
| Final Extension | 72 °C | 5 minutes | |
| Hold | 4 – 10 °C |
Thermocycling Conditions for a Routine PCR