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Phusion Plus DNA Polymerase: PCR Protocol |
This protocol is intended for amplification of DNA fragments by PCR. The Thermo Scientific Phusion Plus DNA Polymerase used in this PCR protocol is well-suited for applications such as high-fidelity PCR, long-range PCR, cloning, mutagenesis, and sequencing.
Materials required
- Template: genomic DNA, plasmid, phage DNA, cDNA
- Phusion Plus DNA Polymerase
- Forward and reverse primers
- 10 mM dNTP Mix
- Thermal cycler such as Proflex PCR System
- 0.2 or 0.5-mL nuclease-free microcentrifuge tubes
- Water, nuclease-free
Contents in Phusion Plus DNA Polymerase Kit
- Phusion Plus DNA Polymerase (50 μL)
- 5X Phusion Plus Buffer (1.25 mL)
- 5X Phusion GC Enhancer (1.25 mL)
Store at –15°C to –25°C.
PCR steps
1. Prepare reaction by adding the following components in the order listed in the following table.
| Component | 20 µL rxn | 50 µL rxn | Final conc. |
|---|---|---|---|
| 5X Phusion Plus Buffer [1] | 4 µL | 10 µL | 1X |
| Forward primer | x μL | x μL | 0.5 µM [2] |
| Reverse primer | x μL | x μL | 0.5 µM [2] |
| 10 mM dNTPs | 0.4 µL | 1 µL | 200 µM each |
| Template DNA | x μL | x μL | 0.01–10 ng plasmid 5–100 ng genomic DNA |
| 5X Phusion GC Enhancer [3] | 4 µL | 10 µL | 1X |
| Phusion Plus DNA Polymerase | 0.2 µL | 0.5 µL | — |
| Water, nuclease free | add to 20 µL | add to 50 µL | — |
Tips:
- Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery.
- Prepare a master mix for the appropriate number of samples to be amplified.
- Pipette polymerase carefully and gently, as the high glycerol content (50%) in the storage buffer may lead to pipetting errors.
- Take precautions to avoid cross-contamination by using aerosol-resistant barrier tips and analyzing PCR products in a separate area from PCR assembly.
Notes:
[1] Provide 1.7 mM MgCl2 at 1X concentration.
[2] Reduce the primer concentration to 0.2 μM final concentration when amplifying >5 kb targets from genomic DNA and for multiplex reactions.
[3] Use only for targets with >65% GC content.
- Run a thermal cycler program set to the following parameters according to the protocol to be performed.
a. 3-step protocol parameter
| Cycle step | Temp. | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 98°C | 30 s | 1 |
| Denaturation Annealing Extension | 98°C 60°C 72°C | 5–10 s 10 s 15–30 s/kb | 25–35 |
| Final extension | 72°C 4°C | 5 min Hold | 1 Hold |
b. 2-step protocol (for primers >30 nt in length)
| Cycle step | Temp. | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 98°C | 30 s | 1 |
| Denaturation Annealing/extension | 98°C 72°C | 5–10 s 15–30 s/kb | 25–35 |
| Final extension | 72°C 4°C | 5 min Hold | 1 Hold |
Phusion Plus DNA Polymerase formats
| Stand-alone enzyme | 2X master mix | |
|---|---|---|
| Phusion Plus DNA Polymerase | Phusion Plus PCR Master Mixes (colorless) | Phusion Plus PCR Master Mixes (green) |
Flexibility to optimize reaction conditions |
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Frequently asked questions (FAQs)
Primer annealing is performed at 60°C because proprietary additives in the reaction buffer stabilize primer—template duplexes during annealing and eliminate the need to optimize annealing temperature for each primer pair.
If preferred, use Tm calculator to calculate primer annealing temperature.
Phusion Plus DNA Polymerase amplifies up to 10 kb from genomic DNA and 20 kb from low complexity DNA.
Use the provided Phusion GC Enhancer for amplicons with >65% GC content.
- Recommended final primer concentration is 0.5 μM and can be varied in a range of 0.1–1.0 μM, if needed. Lower primer concentrations (0.2 μM final) are recommended for amplification of >5 kb targets from high complexity DNA and multiplex reactions.
- Design 18- to 35-mers with 40–60% GC content. Avoid primer pairs with complementarity at 3' ends or >10°C melting temperature (Tm) difference.
- Verify primer complementarity to a single template region using programs for sequence alignment. Online primer design programs such as the Invitrogen OligoPerfect Designer can be helpful.
- Low complexity DNA: Optimal amount of low complexity DNA (plasmid, phage or BAC DNA) is 0.01–10 ng per 50 μL reaction, although it can be varied from 0.1 pg to 50 ng per 50 μL reaction.
- Genomic DNA: Optimal amount of genomic DNA is 5–100 ng per 50 μL reaction, although it can be varied from 0.1–250 ng per 50 μL reaction. A higher template amount is recommended for long targets.
- cDNA: Optimal amount of cDNA is 0.1–1 μL of the first-strand reaction mixture per 50 μL reaction.
The total number of PCR cycles can vary from 15 to 40, depending on the target length and template amount. For low complexity templates 20–25 PCR cycles are typical; 30–35 cycles are recommended for genomic DNA.
Denaturation
- Use 98°C for denaturation. Make sure that the heated lid temperature is set at several degrees above 98°C to avoid sample condensation.
- 30-second initial denaturation at 98°C is sufficient for most templates. The initial denaturation time can be increased up to 5 minutes if necessary.
Annealing
- Due to unique isostabilizing molecules in the reaction buffer, 60°C annealing temperature works for most primers.
- The 2-step protocol is recommended when primers without non-complementary parts are >30 nt in length, e.g., primers for site-specific mutagenesis. In the 2-step protocol, the combined annealing/extension step should be performed at 72°C.
- If amplification does not give satisfactory results, we recommend a temperature gradient. The annealing temperature can be optimized using Applied Biosystems thermal cyclers, such as the ProFlex PCR System or the VeritiPro Thermal Cycler featuring VeriFlex technology.
Extension
- Extension time depends on amplicon length and complexity. For low complexity DNA (e.g., plasmid, phage or BAC DNA) use an extension time of 15 seconds per 1 kb. For high complexity genomic DNA, 30 seconds per 1 kb is recommended.
- The extension step can be prolonged up to 90 sec/kb for targets up to 5 kb without negative effect on specificity. This allows for amplifying shorter and longer amplicons together using the same protocol.
| Observation | Possible cause | Recommended action |
|---|---|---|
| No product or low yield | Sub-optimal cycling parameters |
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| Problem with reaction mix |
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| Observation | Possible cause | Recommended action |
|---|---|---|
| Non-specific products or smeared bands | Sub-optimal cycling parameters |
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| Primer concentration is too high | Reduce primer concentration |
Phusion Plus DNA Polymerase offers several advantages over traditional Phusion DNA polymerases. It eliminates the need for annealing temperature (Tm) calculations by using a universal annealing temperature for all primers and allows co-cycling of targets of different lengths, reducing PCR runs. Additionally, it provides higher PCR sequence accuracy (>100x that of Taq enzyme), higher sensitivity for detecting low-abundance targets, better performance with GC-rich sequences due to a new GC enhancer, and greater tolerance to PCR inhibitors.
For Research Use Only. Not for use in diagnostic procedures.
Stylesheet for Classic Wide Template adjustments