Q5™ Hot Start High-Fidelity DN

Q5 Hot Start High-Fidelity DNA Polymerase is a high-fidelity, thermostable, hot start DNA polymerase with 3´→ 5´ exonuclease 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 DNA Polymerase is ideal for cloning and can be used for long or difficult amplicons. Q5 Hot Start High-Fidelity DNA Polymerase is supplied with an optimized buffer system that allows robust amplification regardless of GC content. The 5X Q5 Reaction Buffer contains 2 mM MgCl₂ at final (1X) reaction concentrations and is recommended for most routine applications. For GC-rich targets (≥ 65% GC), amplification can be improved by the addition of the 5X Q5 High GC Enhancer. Q5 Hot Start High-Fidelity DNA Polymerase is unlike typical, lower fidelity PCR enzymes. To determine the optimal annealing temperatures for a given set of primers, use of the NEB T_mCalculator is highly recommended.







Amplification of a variety of human genomic amplicons from low to high GC content using Q5 Hot Start High-Fidelity DNA Polymerase. 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

Reagents Supplied:
Q5 High GC Enhancer
Q5 Reaction Buffer


Enzyme Properties


Heat Inactivation:
No


Reaction & Storage Conditions



Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C.

Unit Assay Conditions:
25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl₂, 1 mM β-mercaptoethanol, 200 μM dNTPs including [³H]-dTTP and 400 μg/ml activated Calf Thymus DNA.

Reaction Conditions:
1X Q5 Reaction Buffer, DNA template, 0.5 μM primers, 200 μM dNTPs (not included), 1X Q5 High GC Enhancer (optional) and 1 unit of Q5 Hot Start High-Fidelity DNA Polymerase in a total reaction volume of 50 μl.

Concentration:
2,000 units/ml

Storage Temperature:
-20°C


FAQs

1. What are the advantages to using Q5™ High-Fidelity DNA Polymerase?
2. What are the advantages to using Q5™ Hot Start High-Fidelity DNA Polymerase?
3. What is the fidelity of Q5 High-Fidelity DNA Polymerase?
4. How should I determine an appropriate annealing temperature for my reaction?
5. What should my primer concentration be when using Q5 High-Fidelity DNA Polymerase products?
6. How should I set up a PCR reaction using Q5™ Hot Start High-Fidelity DNA Polymerase?
7. My template is GC rich or supercoiled. How can I optimize my product yield using Q5™ High-Fidelity DNA Polymerase?
8. Do I need to modify my annealing temperature when using the Q5™ High GC Enhancer?
9. When should I add the High GC Enhancer?
10. How do I activate Q5™ Hot Start High-Fidelity DNA Polymerase?
11. 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?
12. There is a precipitate in the bottom of the buffer tube. Is this normal?
13. What length of product can be made by Q5™ High-Fidelity DNA Polymerase?
14. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Q5™ High-Fidelity DNA Polymerase?
15. Does Q5™ High-Fidelity DNA Polymerase exhibit a strand displacement activity?
16. Where can I find help troubleshooting my PCR?
17. Will Q5™ High-Fidelity DNA Polymerase incorporate dUTPs?
18. I'd like to clone a fragment amplified with Q5™ High-Fidelity DNA Polymerase. Do I have to blunt-end clone?
19. Do other polymerases work in Q5 Reaction Buffer?

Protocols


Protocols for Q5™ Hot Start High-Fidelity DNA Polymerase


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 1.0 unit of Q5 Hot Start High-Fidelity DNA Polymerase in the presence of 200 μM dNTPs and 0.5 µM of each primer in Q5 Reaction Buffer 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 1.0 unit of Q5 Hot Start High- Fidelity DNA Polymerase in the presence of 200 μM dNTPs and 1.0 μM of each primer in Q5 Reaction Buffer result in the expected 20 kb product.

Enhancer-Dependent High GC (65% GC) PCR:
30 cycles of PCR amplification in a 50 μl reaction containing 20 ng genomic DNA with 1.0 unit of Q5 Hot Start High-Fidelity DNA Polymerase in the presence of 200 μM dNTPs, 1X Q5 High GC Enhancer and 0.5 μM of each primer in Q5 Reaction Buffer result in the enhancer-dependent production of the 452 bp high GC product.

Hot Start-Specific Genomic DNA PCR:
25 cyclesof PCR amplification in a 25 ìl reaction containing50 ng genomic DNA with 0.5 units of Q5 HotStart High-Fidelity DNA Polymerase in the presenceof 200 ìM dNTPs, 1X Q5 High GC Enhancer and0.5 ìM of each primer in Q5 Reaction Buffer resultin the expected 665 bp product, free of non-specificamplification products after pre-incubation at roomtemperature for 1 hour.

Endonuclease Activity:
Incubation of a 50 μl reaction in NEBuffer 2 containing a minimum of 10 units of Q5 High-Fidelity DNA Polymerase with 200 μM dNTPs and 1 μg of supercoiled pUC19 DNA for 4 hours at either 37°C or 72°C results in < 10% conversion to the nicked form as determined by agarose gel electrophoresis.

Physical Purity:
Purified to > 97% homogeneity as determined by SDS-PAGE analysis using Coomassie Blue detection.

qPCR DNA Contamination (E.coli Genomic):
A minimum of 4 units of Q5 High-Fidelity DNA Polymerase is screened for the presence of E. coli genomic DNA using SYBR® Green qPCR with primers specific for the E. coli 16S rRNA locus. Results are quantified using a standard curve generated from purified E. coli genomic DNA. The measured level of E. coli genomic DNA contamination is less than 1 E. coli genome.


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 2X Master Mix
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.