High-fidelity PCR
A bench workflow from reaction planning and master mix setup to post-run verification. Phusion is the worked example; settings must follow the polymerase actually used.
- Document ID
- PC-PRO-001
- Revision
- 1.0
- Content review
- 2026-08-31
- Document type
- Research reference workflow
01Scope and exclusions
Reference workflow for routine DNA amplification with NEB Phusion High-Fidelity DNA Polymerase. Other polymerases, hot-start systems, long or GC-rich templates, and clinical/diagnostic samples require the applicable product manual and institutional validation.
02Critical parameters and conditions
| Parameter | Reference value | Applicability note |
|---|---|---|
| Reaction volume | 20 µL or 50 µL | NEB Phusion example; preserve final concentrations when scaling. |
| Final primer concentration | 0.5 µM each | Equivalent to 1 µL of each 10 µM primer in a 20 µL reaction. |
| Final dNTP concentration | 200 µM each | For a 10 mM dNTP mix, add 0.4 µL to a 20 µL reaction. |
| Cycling framework | 98°C 30 s; 25–35 cycles | Denature at 98°C for 5–10 s; determine annealing with NEB Tm Calculator; extend at 72°C for 15–30 s/kb. Applies only to the cited Phusion conditions. |
03Required controls
- 01No-template control (NTC): monitors contamination in master mix, environment, or pipetting.
- 02Positive amplification control: confirms reaction competence for new primers, template types, or troubleshooting.
- 03Where relevant, include extraction blanks or no-RT controls for signal introduced upstream.
04Reagents, consumables, and equipment
- High-fidelity polymerase, supplied buffer, and dNTPs
- Forward/reverse primers, template DNA, and nuclease-free water
- PCR tubes, ice bucket, pipettes, and thermocycler
- No-template control and, where appropriate, a positive control
05Procedure
Confirm the reaction system
Open the current polymerase manual and record buffer strength, primer concentration, template range, annealing-temperature method, and extension rate. Do not mix parameters across enzymes.
Plan controls and tube positions
Include at least a no-template control and add a positive control when reaction performance must be distinguished from sample failure. Make a loading map before labeling tubes.
Prepare the master mix
Thaw and gently mix components on ice. Prepare a template-free master mix with a sensible pipetting overage; add polymerase last when directed by its manual.
Aliquot and add template
Aliquot the master mix, add templates and controls in a separate area, change tips to prevent cross-contamination, and briefly spin down.
Run the thermocycler
For Phusion, the routine reference uses 98°C initial denaturation, 25–35 cycles, annealing determined with the NEB Tm tool, and roughly 15–30 s/kb extension depending on template complexity. Other enzymes require their own settings.
Verify and document
Choose agarose gel analysis, fragment analysis, or cleanup according to the goal. Inspect controls before interpreting samples, and record the program, template input, primer lots, and anomalies.
06Acceptance criteria
- No visible NTC amplification at the target size; the positive control yields the expected band.
- The principal sample band matches expected size and nonspecific products or smearing are acceptable for the downstream use.
- All deviations, reagent substitutions, and repeats are documented.
07Required records
- Template ID, type, input amount, and storage location
- Primer sequences/lots, working concentration, and target length
- Polymerase catalog/lot, reaction recipe, cycling program, and original gel image
Critical boundary
Temperatures and times on this card apply only to the cited Phusion example. Polymerase choice, template GC content, and amplicon length change the optimum.
Safety note
Separate post-amplification work from reaction setup. Handle gel stains, UV/blue-light imaging, and sample waste under laboratory SDS, SOP, and zoning controls.