This standard PCR protocol is intended for amplification of DNA fragments using Invitrogen Platinum SuperFi II DNA Polymerase, which is a proofreading DNA polymerase that combines high fidelity with Platinum hot-start technology and universal primer annealing. It is an ideal choice for cloning, mutagenesis, and other applications.

Due to proprietary additives in the reaction buffer, Platinum SuperFi II DNA Polymerase shows efficient amplification of both AT and GC rich targets. Additional DNA melting agents are not required for GC-rich PCR (up to 75% GC).

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Materials required

Contents in Platinum SuperFi II PCR Polymerase package

  • Platinum SuperFi II PCR Polymerase
  • 5X SuperFi II Buffer

Storage: Store all components at –20°C
Kit sizes: 100 reactions, 500 reactions, 2500 reactions

Ordering information

PCR steps

1. Thaw, mix, and briefly centrifuge each component before use.
2. Prepare PCR reaction mix by adding the following components to each PCR tube. Mix and centrifuge components. 

ComponentFinal concentration20-µL rxn50-µL rxn
Water, nuclease-free—to 20 µLto 50 µL
5X SuperFi II Buffer1X4 µL10 µL
10 mM dNTPs200 µM each0.4 µL1 µL
Platinum SuperFi II DNA Polymerase—0.4 µL1 µL

3. Add template DNA and primers to each tube for a final reaction volume of 20-µL or 50-µL.

ComponentFinal concentration20-µL rxn50-µL rxn
10 µM forward primer0.5 µMx µLx µL
10 µM reverse primer0.5 µMx µLx µL
Template DNA0.1–10 ng plasmid DNA (5–100 ng genomic DNA)x µLx µL
Tips:
  • For multiplex reactions and for amplification of targets >5 kb from genomic DNA, reduce the final primer concentration to 0.2 μM.
  • Store enzymes at –20°C and avoid repeated freeze–thaw cycles to preserve activity.
  • Use 0.1–10 ng plasmid DNA or 5–100 ng genomic DNA per 50 µL reaction.
  • Recommended primer concentration:
    • Standard: 0.5 µM final concentration
    • Long (>5 kb) or multiplex targets: 0.2 µM final concentration
Notes:
  • Mg2+: Platinum SuperFi II Reaction Buffer provides MgCl2 at a final concentration of 1.75 mM in the reaction. If the primers and/or the template contain chelators such as EDTA or EGTA, the apparent Mg2+ optimum may be shifted to higher concentrations.
  • DMSO: Platinum SuperFi II DNA Polymerase can amplify targets with high GC content (up to 75% GC) without any additional DNA melting agents. In cases of extremely GC-rich targets (>75% GC), we recommend addition of DMSO to a final concentration of 5%.

4. Run a thermal cycler program set to the following parameters according to the protocol to be performed. Total amount of PCR cycles can vary from 15 to 40, depending on target length and template amount.

  • Low-complexity templates 25–30 PCR cycles
  • Genomic DNA 30–35 cycles

a. 3-step protocol

Cycle stepTemp.TimeCycles
Initial denaturation98°C30 s1
Denaturation
Annealing
Extension 		98°C
60°C
72°C		5–10 s
10 s
15–30 s/kb		25–35
Final extension72°C
4°C		5 min
Hold		1
Hold

b. 2-step protocol (for primers >30 nt length)

Cycle stepTemp.TimeCycles
Initial denaturation98°C30 s1
Denaturation
Annealing/extension		98°C
72°C		5–10 s
15–30 s/kb		25–35
Final extension72°C
4°C		5 min
Hold		1
Hold
Tips:
  • Denaturation: Use 98°C for denaturation. Ensure that the heated lid temperature is set several degrees above 98°C to avoid sample condensation.
  • Annealing: Due to unique iso-stabilizing molecules in the Platinum SuperFi II Reaction Buffer, 60°C annealing temperature works for most primers.
  • Extension:
    • For low-complexity DNA (e.g., plasmid, lambda or BAC DNA), use an extension time of 15 seconds per 1 kb.
    • For high complexity genomic DNA, use an extension time of 30 seconds per 1 kb.
    • For targets up to 5 kb, prolong extension step up to 90 seconds per 1 kb without negative effect on specificity.

5. Use PCR product immediately in downstream application or store at –20°C.

SuperFi II DNA Polymerase formats

Stand-alone enzyme2X master mix
Platinum SuperFi II DNA PolymerasePlatinum SuperFi II PCR Master Mixes (colorless)Platinum SuperFi II PCR Master Mixes (green)

Flexibility to optimize reaction conditions

Learn more

  • Convenient ready-to-use 2X mixture includes all necessary PCR components except the template and primers
  • Simple reaction setup with minimal pipetting steps

Learn more
Download protocol

  • Convenient ready-to-use 2X mixture includes all necessary PCR components except the template and primers
  • Simple reaction setup with minimal pipetting steps
  • Contains two tracking dyes and a density reagent for direct loading of PCR products on gels

Learn more
Download protocol

FAQs on PCR protocol using Platinum SuperFi II DNA Polymerase

What are the advantages of Platinum SuperFi II DNA Polymerase over Taq DNA polymerases?

Platinum SuperFi II offers >300× higher fidelity and faster extension rates than Taq based DNA polymerases. Additionally, the antibody-based hot-start system keeps the enzyme inactive at room temperature, preventing nonspecific amplification until the initial denaturation step.

How is the hot-start PCR protocol different from the standard PCR protocol?

There is no difference in the PCR protocol steps themselves between a standard PCR protocol and a hot-start PCR protocol. Both follow the same thermal cycling stages—denaturation, annealing, and extension. The key difference lies in when the DNA polymerase becomes active during the reaction.

Standard PCR protocol: The DNA polymerase is active during reaction setup at room temperature, which can lead to primer–dimer formation or nonspecific amplification before thermal cycling begins.

Hot-start PCR protocol: The DNA polymerase remains inactive during setup and is activated only after an initial high-temperature denaturation step, reducing nonspecific products and improving reproducibility.

Because of this controlled enzyme activation, the hot-start PCR protocol is preferred for high-fidelity PCR, amplification of difficult or low-copy templates, multiplex PCR, and high-throughput workflows, where specificity and consistency are critical.

What is the maximum amplicon length achievable with Platinum SuperFi II DNA Polymerase?

It can amplify targets up to 20 kb from plasmid templates under optimal conditions.

Do I need to add DMSO or GC enhancers for GC-rich targets?

No, the enzyme efficiently amplifies templates up to 75% GC without additional agents; for >75% GC, add up to 5% DMSO.

How much template DNA should be used?
  • Low-complexity DNA: Optimal amount of low-complexity DNA (plasmid, phage or BAC DNA) is 0.1–10 ng per 50 μL reaction, but it can be varied from 0.1 pg to 50 ng per 50 µL reaction. For long targets, we recommend using higher amounts of template.
  • Genomic DNA: Optimal amount of genomic DNA is 5–100 ng per 50 μL reaction, but it can be varied from 0.1 ng to 250 ng per 50 μL reaction. We recommend higher template amount for long targets.
  • cDNA: Optimal amount of cDNA is 0.1–1 μL of the first-strand reaction mixture per 50 μL reaction.
What are the recommended primer optimizations for successful PCR?
  • Design 18–35-mers with 40–60% GC content. If possible, design the primers with one or two G or C bases at the 3’ end. 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 primer design tool can be helpful.
  • We recommend a final primer concentration of 0.5 μM, but this can be varied in a range of 0.1–1.0 μM, if needed. For amplification of >5 kb targets from high complexity DNA and for multiplex reactions, we recommend lower primer concentrations (0.2 μM final).
What are the recommended PCR cycling parameters?
  • Denaturation
    • Use 98°C for denaturation.
    • Ensure that the heated lid temperature is set several degrees above 98°C to avoid sample condensation.
    • 30-second initial denaturation at 98°C is sufficient for most templates. You can extend the initial denaturation time up to 5 minutes, if needed.
       
  • Annealing
    • Due to unique iso-stabilizing molecules in the Platinum SuperFi II Reaction Buffer, 60°C annealing temperature works for most primers.
    • The 2-step protocol when primers without non-complementary parts are >30 nt in length (e.g., primers for site-specific mutagenesis). In the 2-step protocol, perform the combined annealing/extension step at 72°C. If the amplification does not give satisfactory results, we recommend using a temperature gradient. The annealing temperature can be optimized using Applied Biosystems thermal cyclers, such as the ProFlex PCR System or the Veriti Thermal Cycler featuring VeriFlex technology.
       
  • Extension: Extension time depends on amplicon length and complexity:
    • For low-complexity DNA, such as plasmid, lambda, or BAC DNA, use an extension time of 15 seconds per 1 kb.
    • For high-complexity genomic DNA, use an extension time of 30 seconds per 1 kb.
    • You can prolong the extension step up to 90 seconds per 1 kb for targets up to 5 kb without negative effect on specificity. Prolonged extension time allows the amplification of shorter and longer amplicons together using the same protocol.
What is the advantage of universal primer annealing feature of the enzyme?

Primer annealing is a critical step in the polymerase chain reaction (PCR), during which primers bind to the flanking sequences of the target DNA to enable amplification. Typically, the annealing temperature must be carefully calculated and optimized based on the melting temperature (Tm) of each primer pair to ensure specific binding and efficient amplification.

The innovative buffer formulation of Platinum SuperFi II DNA Polymerase enables reliable primer annealing at a universal temperature of 60°C, irrespective of primer sequence. This eliminates the need for extensive annealing temperature optimization while maintaining high specificity and yield.

This universal primer annealing feature also simplifies co-cycling assays, allowing multiple targets to be amplified in the same run without significant optimization.

Learn more about universal annealing

What should I do if no PCR product or low yield is observed?

Recommended actions:

  • Repeat the PCR and make sure that there are no pipetting errors.
  • Use fresh, high-quality dNTPs. Do not use dNTP mix or primers that contain dUTP or dITP.
  • Check primer design and concentration.
  • Run a temperature gradient to determine optimal annealing temperature.
  • Increase the total number of cycles.
  • Titrate the template amount. Too little or too much template can compromise PCR results.
What should I do if nonspecific or smeared bands appear?

Recommended actions:

  • Run a temperature gradient to determine optimal annealing temperature.
  • Decrease extension time.
  • Reduce the total number of cycles.
  • Reduce the primer concentration.

For Research Use Only. Not for use in diagnostic procedures.

Stylesheet for Classic Wide Template adjustments