Q5™ Hot Start High-Fidelity 2X

The Q5 Hot Start High-Fidelity 2X Master Mix features a high-fidelity, thermostable hot start DNA polymerase with 3´→ 5´ exo-nuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. The addition of an aptamer-based inhibitor allows room temperature reaction setup. With an error rate > 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase, Q5 Hot Start High-Fidelity 2X Master Mix is ideal for cloning and can be used for long or difficult amplicons. The convenient master mix formulation is supplied at a 2X concentration. The mix contains dNTPs, Mg⁺⁺ and a proprietary broad-use buffer requiring only the addition of primers and DNA template for robust amplification regardless of GC content. When used at the recommended 1X final concentration, the Q5 Hot Start High-Fidelity Master Mix contains 2 mM MgCl₂. Q5 Hot Start High-Fidelity 2X Master Mix is unlike typical, lower fidelity PCR enzymes. To determine the optimal annealing temperatures for a given set of primers, use of the NEB T_m Calculator is highly recommended.




Reaction Conditions:
1X Q5 Hot Start High-Fidelity Master Mix, DNA template and 0.5 μM primers in a total reaction volume of 50 μl.





Amplification of a variety of human genomic amplicons from low to high GC content using Q5 Hot Start High-Fidelity 2X Master Mix. All reactions were set up at room temperature, conducted using 30 cycles of amplification and visualized by microfluidic LabChip® analysis.



Source:
An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

Applications:

• High-specificity PCR
• High-fidelity PCR
• Cloning
• Long or Difficult Amplification
• High-throughput PCR

Enzyme Properties


Heat Inactivation:
No


Reaction & Storage Conditions



Concentration:
2 X

Storage Temperature:
-20°C


Notes


General notes:

1. A precipitate (most noticeable after the first 1–2 freeze/thaw cycles) is not uncommon. To ensure optimal performance, the master mix should be thawed and resuspended prior to use. Stability testing using up to 15 freeze/thaw cycles has shown no negative effect on master mix performance.
2. Product specifications for individual components in the Q5 Hot Start High-Fidelity 2X Master Mix are available separately.

FAQs

1. What are the advantages to using Q5™ Hot Start High-Fidelity 2X Master Mix?
2. What is the fidelity of Q5 High-Fidelity DNA Polymerase?
3. How should I determine an appropriate annealing temperature for my reaction?
4. What should my primer concentration be when using Q5 High-Fidelity DNA Polymerase products?
5. How should I set up a PCR reaction using Q5™ Hot Start 2X Master Mix?
6. My template is GC rich or supercoiled. How can I optimize my product yield using Q5™ High-Fidelity Master Mixes?
7. There is a precipitate in the bottom of the Master Mix tube? Is this normal?
8. How do I activate Q5™ Hot Start High-Fidelity DNA Polymerase?
9. Are the DNA fragments produced by Q5™ High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields?
10. What length of product can be made by Q5™ High-Fidelity DNA Polymerase?
11. I have a tube of the Q5™ High GC Enhancer from another product formulation - can I add it to the Q5 Master Mix?
12. What are the stability and storage requirements of the Q5™ Master Mixes?
13. Does Q5™ High-Fidelity DNA Polymerase exhibit a strand displacement activity?
14. Where can I find help troubleshooting my PCR?
15. Will Q5™ High-Fidelity DNA Polymerase incorporate dUTPs?
16. I'd like to clone a fragment amplified with Q5™ High-Fidelity DNA Polymerase. Do I have to blunt-end clone?

Protocols


Protocols for Q5™ Hot Start High-Fidelity 2X Master Mix


Quality Control for Current Lot


Quality control values for a specific lot can be found on the datacard which accompanies each product.

7 kb Genomic DNA PCR:
30 cycles of PCR amplification in a 50 μl reaction containing 20 ng genomic DNA with 1X Q5 Hot Start High-Fidelity Master Mix and 0.5 μM of each primer result in the expected 7 kb product.

20 kb Lambda DNA PCR:
22 cycles of PCR amplification in a 50 μl reaction containing 10 ng Lambda DNA with 1X Q5 Hot Start High-Fidelity Master Mix and 1.0 μM of each primer result in the expected 20 kb product.

Hot Start-Specific Genomic DNA PCR:
25 cycles of PCR amplification in a 25 μl reaction containing 50 ng genomic DNA with 1X Q5 Hot Start High-Fidelity Master Mix in the presence of 0.5 μM of each primer result in the expected 665 bp product, free of non-specific amplification products after pre-incubation at room temperature for 1 hour.


Companion Products


Deoxynucleotide Solution Mix
Deoxynucleotide Solution Set
Magnesium Chloride (MgCl₂) Solution
Q5™ High-Fidelity 2X Master Mix
Q5™ High-Fidelity DNA Polymerase
Q5™ Hot Start High-Fidelity DNA Polymerase
Q5™ Reaction Buffer Pack


Legal


Licenses/Patents/Disclaimers:
This product is licensed from Bio-Rad Laboratories, Inc. under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645 and corresponding patents in other countries for use only in: (a) standard (non-real time) PCR in the research field only, but not real-time PCR or digital PCR; (b) any in-vitrodiagnostics application, except for applications using real-time or digital PCR; and (c) any non-PCR applications in DNA sequencing, isothermal amplification and the production of synthetic DNA.