Invitrogen Platinum SuperFi DNA Polymerase
Invitrogen Platinum SuperFi DNA Polymerase is a proofreading DNA polymerase which combines superior fidelity with trusted Platinum hot-start technology designed for the highest success in PCR. Featuring >100x Taq fidelity, Platinum SuperFi DNA Polymerase is ideally suited for cloning, mutagenesis, and other applications benefiting from supreme sequence accuracy.
- Exceptional >100x Taq fidelity
- High specificity and increased yields with Platinum hot-start technology
- Robust amplification of difficult-to-amplify targets including those of suboptimal purity or with ˃65% GC content
- Convenient workflow with room temperature reaction setup and direct gel loading with green buffer formats
Platinum SuperFi DNA Polymerase offers the highest level of confidence for preserving DNA sequence accuracy with its extremely low error rate. The relative fidelity values of Platinum SuperFi DNA Polymerase and other DNA polymerases were determined using next-generation sequencing. The relative fidelity of Platinum SuperFi DNA polymerase was calculated to be >100x that of Taq DNA polymerase.
Relative fidelity values of different DNA polymerases. Polymerase fidelity was measured by next-generation sequencing. The background level of experimental errors was estimated from PCR-free library sequencing data. Due to the very low error rates associated with the high-fidelity enzymes, it is challenging to quantify statistically significant differences; however, reactions were run in parallel and polymerase fidelities normalized to Taq polymerase.
Platinum SuperFi DNA Polymerase amplifies a broad range of sequence content and amplicon lengths with high specificity and yields due to robustness of the enzyme and superior hot-start technology. The Platinum hot-start technology is based on proprietary antibodies that inhibit enzyme activity until the initial PCR denaturation step, preventing non-specific amplification and primer degradation. Hot-start PCR results in a greater yield of the target amplicon with the added convenience of room-temperature PCR setup
Platinum SuperFi DNA Polymerase DNA Polymerase provides high specificity and robust yields. Seven fragments, 500 to 800 bp in length and of varying GC content were amplified from 50 ng of human gDNA. SuperFi GC enhancer was added for 70% and 76% GC fragments. The molecular weight marker is Thermo Scientific ZipRuler Express DNA Ladder 2.
Versatility across broad range of amplicon lengths. Platinum SuperFi DNA Polymerase (far left panel) provides high specificity and yields across range of human gDNA fragments from 0.2 to 20 kb. The same targets were also amplified using competitor DNA polymerases (A—KAPA HiFi HotStart PCR Kit, B—KOD Hot Start, C—PrimeSTAR GXL, and D—PfuUltra II Fusion Hot Start). The molecular weight marker is ZipRuler Express DNA Ladder 2.
The high sensitivity of Platinum SuperFi DNA Polymerase enables detection of low-abundance DNA templates with accurate results. High sensitivity is advantageous in experiments where there is a limited amount of starting material or the target DNA is in low concentration in the sample.
High sensitivity and reliable amplification from low amounts of input DNA. Amplification of 2 kb fragment from 0.4, 2, 10, 50, and 250 ng human gDNA in 50 μL PCR reactions using Platinum SuperFi DNA Polymerase and competitor DNA polymerases (A—KOD Hot Start, B—PfuUltra II Fusion Hot Start, C—KAPA HiFi HotStart PCR Kit, D—HotStar HiFidelity DNA Polymerase Kit, E—Herculase II Fusion, F—Expand High FidelityPLUS PCR System, and G—Advantage HD Polymerase Mix). The estimated copy number is ~100 copies per 0.4 ng of human genomic DNA. The molecular weight marker is ZipRuler Express DNA Ladder 2.
All Platinum SuperFi DNA polymerase formats are supplied with a separate vial of SuperFi GC Enhancer formulated for specific amplification and improved yields of targets with high-GC content.
Enhanced amplification of GC-rich targets. Platinum SuperFi DNA Polymerase provides high specificity and yields of difficult GC-rich targets (top and bottom, far left panels). The grey triangles indicate increasing GC content (%) of the amplified DNA fragments (66, 69, 73, and 76%). The same amplicons were also amplified using competitor DNA polymerases according to manufacturers‘ recommended protocols for GC-rich PCR (A—KAPA HiFi HotStart PCR Kit, B—Q5 Hot Start High-Fidelity, C—Pwo SuperYield, D KOD Hot Start, E—PrimeSTAR GXL and F—PfuUltra II Fusion Hot Start). The molecular weight marker is ZipRuler Express DNA Ladder 2.
Platinum SuperFi DNA Polymerase is engineered with a DNA-binding domain resulting in high processivity and increased resistance to common PCR inhibitors such heparin, xylan, and humic acid.
Resistance to inhibitors. Amplification of a 2 kb human gDNA fragment using Platinum SuperFi DNA Polymerase or competitor high-fidelity DNA polymerases (A—Q5 Hot Start High-Fidelity, B—KAPA HiFi HotStart PCR Kit, C—KOD Hot Start, and D—PrimeSTAR GXL) in reaction mixtures containing 1—no inhibitor, 2—heparin (0.15 µg/µl), 3—xylan (0.5 µg/µl), or 4—humic acid (0.5 ng/µl).The molecular weight marker is ZipRuler Express DNA Ladder 2.
Due its high processivity and extremely low error rate, Platinum SuperFi DNA Polymerase is ideal for accurately amplifying long fragments (up to 20 kb) with high yields and specificity. Recommend PCR cycling conditions for amplification of targets >10 kb are provided in the protocol. Amplification of even larger fragment sizes (up to 40 kb) is possible, but may require additional optimization of reaction conditions and primer design.
Amplification of long fragments. Platinum SuperFi DNA Polymerase (lane P) successfully amplifies 15 kb and 30 kb targets from human genomic DNA (gDNA) and (50 ng DNA in 50 μL PCR reactions). Competitor DNA polymerases were also tested using the same primer sets designed to target long amplicons (A—Q5 Hot Start High-Fidelity, B—KAPA HiFi HotStart PCR Kit, C—KOD Hot Start, D—PrimeSTAR GXL and E—PfuUltra II Fusion HotStart). The molecular weight marker is ZipRuler Express DNA Ladder 2.
Extended stability of the Platinum SuperFi DNA Polymerase enzyme at room temperature enables high-throughput applications.
Platinum SuperFi DNA Polymerase stability at room temperature. PCR reactions were set up and incubated at room temperature 0 and 24 h before loading in the thermal cycler. Even after 24 h of room temperature incubation, Platinum SuperFi DNA Polymerase enables high specificity and yields with its superior hot-start technology. Amplification of a 501 bp human gDNA fragment (50 ng DNA in 50 μL PCR reactions) was performed using Platinum SuperFi DNA Polymerase (lane P) and competitor DNA polymerases (A—Q5 Hot Start High-Fidelity, B—PrimeSTAR HS, C—KAPA HiFi HotStart PCR Kit and D—KOD Hot Start). The molecular weight marker is ZipRuler Express DNA Ladder 2.
Platinum SuperFi DNA polymerase products with green buffer formats offer the convenience of direct gel loading of PCR products. The green buffer contains a density reagent and two tracking dyes, eliminating tedious dye addition steps prior to gel loading. The green buffer is compatible with downstream applications including DNA sequencing, ligation and restriction digestion.
Simplified PCR workflow. The green buffer enables direct gel loading of PCR products, eliminating tedious steps of dye addition. Left lane of gel image shows reaction mixture with green buffer prior to electrophoresis. Right lanes of gel show yellow and blue dye migration following 5 and 15 minutes of electrophoresis.
The optimal annealing temperature for your primers may differ significantly in PCR amplification with Platinum SuperFi DNA Polymerases in comparison to Taq-based polymerases. Always use the Tm calculator (thermofisher.com/tmcalculator) to determine your primers’ Tm values and recommended annealing temperature.
The SuperFi GC Enhancer has been designed for improved amplification of >65% GC sequences. We recommend adding SuperFi GC Enhancer to a concentration of 20% in the final reaction. The detailed instructions are provided in the product manual.
Good lab practices are important for long fragment amplification. These include using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Optimization steps to consider include decreasing denaturation and extension temperatures, lengthening extension times as recommended in the protocols, and increasing template amounts.
Platinum SuperFi DNA Polymerase cannot read dUTP-derivatives or dITP in DNA templates, so the use of these analogues is not recommended. Platinum SuperFi DNA Polymerase can incorporate 7-deaza-dGTP and radiolabeled dNTPs.
The tracking dyes (a blue and a yellow dye) in the green master mix and green PCR buffer formats for the Platinum SuperFi DNA Polymerase stand-alone enzyme have no detectable effects on PCR efficiency or enzyme characteristics.
The tracking dyes (a blue and a yellow dye) in green master mix and green PCR buffer formats for the Platinum SuperFi DNA Polymerase stand-alone enzyme are compatible with downstream applications such as fluorescent automatic DNA sequencing, ligation, and restriction digestion. For applications that require analysis of PCR products by absorbance or fluorescence excitation, we recommend using colorless versions of the products (Platinum SuperFi DNA Polymerase or Platinum SuperFi PCR Master Mix) or purify the PCR products prior to analysis.
Platinum SuperFi DNA Polymerase produces blunt-end PCR products that can be cloned directly into blunt-end cloning vectors. TA cloning is also possible if 3′ dA-overhangs are added after PCR. Purify PCR products of Platinum SuperFi DNA Polymerase before adding the overhangs. The procedure for adding 3′ dA-overhangs (TA cloning) includes the following steps:
1) Purify the PCR product (e.g. with a PCR purification kit or phenol extraction and DNA precipitation). Before adding the overhangs, PCR product must be purified, as the strong proofreading activity of any remaining Platinum SuperFi DNA Polymerase will degrade the added 3′ dA-overhangs.
2) Perform 3′ dA addition with a Taq DNA polymerase.
- Purified PCR product
- 0.2 mM dATP
- 1x Taq Buffer
- 1 U Taq DNA polymerase
- Incubate the reaction for 20 minutes at 72°C.
3) Proceed to TA cloning. For optimal ligation efficiency, we recommend using fresh PCR products, since 3´ dA-overhangs can be lost during storage
Annealing temperature rules for Platinum SuperFi DNA Polymerase are different from many common DNA polymerases (such as Taq DNA polymerases). For optimal results use Tm calculator on our website.
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All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Q5 is a trademark of New England BioLabs, Inc. Pwo and Expand are trademarks of Roche Molecular Systems Inc. PfuUltra and Herculase are trademark of Agilent, Inc. KAPA HiFi HotStart is a trademark of KAPA Biosystems, Inc. HotStar HiFidelity is a trademark of Qiagen, Inc. PrimeSTAR and Advantage are trademarks of Takara- Clontech Laboratories, Inc.