Described here is the protocol for a polymerase chain reaction using Q5 DNA polymerase. The information is largely based on NEB recommendations. –Shyam Bhakta
Reaction Setup
| Component | 25 µL Rxn | 50 µL Rxn | Final Concentration |
|---|---|---|---|
| 5× Q5 Reaction Buffer | 5 µL | 10 µL | 1× |
| 10 mM dNTPs | 0.5 µL | 1 µL | 200 µM |
10 µM Forward Primer or 100 µM | 1.25 µL or 0.15 µL | 2.5 µL or 0.25 µL | 0.5 µM |
10 µM Reverse Primer or 100µM | 1.25 µL or 0.15 µL | 2.5 µL or 0.25 µL | 0.5 µM |
| Template DNA | variable | variable | 1 ng–1 pg plasmid/viral. 1 ng–1 µg genomic. |
| Q5 Polymerase | 0.25 µL (½ U) | 0.5 µL (1 U) | 0.02 U/µL, 1% with 2 U/µL enzyme. |
| 5× Q5 High GC Enhancer (opt) | (5 µL) | (10 µL) | (1×) |
| Nuclease-Free Water | to 25 µL | to 50 µL | - |
- Assemble all reaction components on ice, unless using Q5 Hot-Start Polymerase.
- Gently mix the reaction.
- Collect all liquid to the bottom of the tube by a quick spin if necessary.
- Quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98°C).
Thermocycling Conditions
| Step | Temperature | Time | Notes | |
|---|---|---|---|---|
| Initial Denaturation | 98°C | 30 s–3 min | 30 s for most templates (plasmid/linear/E. coli). 1–3 min for complex, or to better lyse cells. | |
| 25–35 cycles | Denaturation | 98°C | 5–10 s | Not >10 s. |
| Annealing | *50–72°C | 10–30 s | Find Tanl, 3° above Tm of lower Tm primer | |
| Extension | 72°C | 10–30 s/kb | 10–15 s/kb for simple plasmid/E. coli template. 20–40 s/kb for complex genomic/cDNA template. 30–40 s/kb for >6 kb amplicons. | |
| Final Extension | 72°C | 2 min | ||
| Hold | 4–10°C | ∞ |
Troubleshooting/optimizing a PCR
- Add GC-enhancer or other additives. See Additives section.
- Use the touchdown thermocycling protocol. See Annealing section.
- Try different annealing temperatures. See Annealing section.
If you need to destroy the non-synthetic template (plasmid), you can add 1 µL DpnI per 50 µL completed PCR, and incubate 37˚C, 15–60 min.
Detailed Guidelines
Template
Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template are:
| DNA | Concentration |
|---|---|
| DNA Genomic | 1 ng–1 µg per 50 µL PCR |
| Plasmid or Viral | 1 pg–1 ng per 50 µL PCR |
Primers
Oligonucleotide primers are generally 17–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (in Benchling) can be used to design or analyze primers. The best results typically come from reactions with 0.5 µM each primer.
Buffers
The 5× Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5× Q5 Reaction Buffer is detergent-free and contains 2.0 mM MgCl2 at the final (1×) concentration. Shyam deduced that Q5 Reaction Buffer contains glycerol, which reduces DNA secondary structure, and tetramethylammonium, which increases primer stringency.
Additives
Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1× Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.
Mg2+ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1×, the Q5 Reaction Buffer provides the optimal Mg2+concentration.
Shyam deduced that Q5 buffer contains glycerol, which reduces DNA secondary structure, and tetramethylammonium, which increases primer stringency.
- DMSO reduces DNA secondary structure. Use at 3–10% final. 10% DMSO lowers Tm 5.5–6°C. DMSO reduces the activity of Taq polymerase.
- Glycerol reduces DNA secondary structure. Use at 5–10% final.
- Formamide increases the stringency of primer annealing, resulting in less non-specific priming and increased amplification efficiency. Use at 1-10%.
- Tetramethylammonium increases the stringency of primer annealing, resulting in less non-specific priming and increased amplification efficiency. Use at 10–100 mM final.
- Triton X-100, Tween 20 or NP-40 reduce DNA secondary structure, but can increase non-specific amplification. Use at 0.1–1% final.
- Betaine / Betaine·H₂O greatly reduces the high Tm bias of G:C over A:T pairs, reversing the bias slightly at lower Tms. Especially useful for GC-rich templates. Use at 1–3 M final. Can inhibit amplification of some templates. Don't use betaine·HCl.
- 1,2-propanediol greatly improves success of GC-rich template amplification. Use at 0.8 M final.
- Ethylene glycol greatly improves success of GC-rich template amplification. Use at 1 M final.
- 7-deaza-2′-deoxyguanosine is a dGTP analogue especially useful for extremely GC-rich templates. Success is reported with up to 83% GC. Use a 1:3 ratio of dGTP:7-deaza-2′-deoxyguanosine.
- BSA prevents reaction components adhering to the tube. Use at ≤0.8 mg/mL final.
- Tween-20/-40 can neutralize SDS left over from template DNA preparation that would inhibit the reaction. Use at 0.25–1% final.
Sources: Bitesize Bio 1, Bitesize Bio 2, Bitesize Bio 3
Deoxynucleotides
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.
Q5 DNA Polymerase concentration
We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 U/mL (1.0 U/50 μl reaction). However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40 U/mL (0.5–2 U/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 U/50 μl reaction, especially for amplicons longer than 5 kb.
The hot-start formulation, Q5 Hot-Start DNA Polymerase, inhibits the robust exonuclease (and polymerase?) activity of the enzyme, allowing for convenient room temperature reaction setup. The aptamer/inhibitor is released from the enzyme during normal cycling conditions, so no separate activation step is required.
Denaturation
An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer 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.
Annealing
Optimal annealing temperatures for Q5 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. Typically, use a 10–30 second annealing step at 3°C above the Tm of the lower Tm primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
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.
Extension
The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.
A final extension of 2 minutes at 72°C is recommended.
Cycle number
Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.
PCR product
The PCR products generated using Q5 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 Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated.