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Polymerase Chain Reaction (PCR)

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High-Fidelity PCR Enzymes

Successful polymerase chain reaction (PCR) is crucial for amplifying DNA sequences in order to study their function, either by sequencing, mutation, transcription, or expression of gene products. PCR involves replication of the DNA template by a thermostable DNA polymerase. The processivity, specificity, and fidelity of the polymerase enzyme used can largely determine the efficiency, reproducibility, and yield of the PCR reaction.

MilliporeSigma’s molecular biologists work to develop the newest, best performing polymerases for customer use, optimizing conditions, buffer compositions, and cycling parameters to save you valuable time and resources.

Recent CRISPR Publications Using KOD Hot Start

Genome Engineering with CRISPR-Cas9 in the Mosquito Aedes aegypti. Cell. 2015 Apr 7;11(1):51-60.

Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development. 2014 Dec;141(24):4827-30.

CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nature Protocols. 2014 Oct;9(10):2493-512.

“We started to use KOD Xtreme Hot Start DNA polymerase for long range PCR when screening ES cells. We found that it is more robust at amplifying targets > 3kb in length and produces cleaner, more interpretable bands than other polymerases.”

University of Connecticut, Gene Targeting and Transgenic Facility

Choose the Appropriate Thermostable Polymerase for PCR Applications:

Enzyme
KOD DNA Polymerase
KOD Hot Start DNA Polymerase
KOD XL DNA Polymerase
KOD Xtreme™ Hot Start DNA Polymerase
PCR Product Size (kb) <6 <21 <30 <40
Applications Cloning, cDNA amplification Cloning, cDNA amplification Crude samples, multiplex, incorporation of derivatized dNTPs Crude samples, long targets, difficult and GC-rich targets
  • KOD-XL DNA polymerase amplification results in a mixture of blunt and 3’-dA products while other KOD DNA polymerases generate blunt end products.
  • NovaTaq™ is also available for routine PCR.

KOD DNA Polymerase Is a Highly Efficient Proof Reading Enzyme.

Enzyme
KOD DNA Polymerase
Pfu DNA Polymerase
Taq DNA Polymerase
Species Thermococcus kodakaraensis Pyrococcus furiosus Thermus aquaticus YT-1
Fidelity† (mutation frequency) 0.0035 0.0039 0.013
Elongation rate (bases/second) 106-138 25 61
Processivity (nucleotide bases) >300 <20 not determined
  • † Fidelity was measured by the authors as mutation frequency in PCR products using a sensitive blue/white phenotypic assay with a 5.2-kb lacZ plasmid as template.

KOD Hot Start DNA Polymerase

This enzyme provides the highest accuracy, yield and processivity of our proofreading DNA polymerases. High processivity provides more product, enabling quick, reliable gene amplification. KOD Hot Start polymerase amplifies genomic DNA up to 12 kbp and plasmid DNA up to 21 kbp, including GC-rich regions. The polymerase is also available as mastermix for high throughput and standardization.
Cycle Profile A
Cycle Profile B
Cycle Profile C
Cycle Profile D
Initial denaturation 98°C 30 s 94°C 2 min 95°C 2 min 95°C 2 min
29 cycles 8°C 10 s 55°C 20 s 72°C 30 s 94°C 15 s 52°C 20 s 68°C 60 s 95°C 20 s 55°C 20 s 72°C 30 s 95°C 20 s 55°C 10 s 70°C 15 s
Final extension 72°C 5 min 68°C 5 min 72°C 3 min N/A

KOD Xtreme™ Hot Start DNA Polymerase

Use this optimized PCR system to reliably amplify long or challenging DNA templates (up to 90% GC content). Ideal for amplifying crude samples with minimal sample preparation, this kit translates into less reagents and less time spent on PCR.
KOD polymerase yields more product in fewer cycles compared to other PCR enzymes.
KOD polymerase yields more product in fewer cycles compared to other PCR enzymes.
Yields were determined by PicoGreen analysis after 19, 21, 23, 25, 27, and 29 cycles for all 4 cycling profiles. The best yield data for each enzyme, from any cycling profile, were graphed. The cycling profile that gave the best yield is identified in parentheses. The shaded area highlights yields in cycles 19-25, which are most preferred for cloning. KOD-HS DNA polymerase out-performed the competition.
KOD Xtreme™ Hot Start Polymerase amplifies GC-rich targets more efficiently than other polymerases.
KOD Xtreme™ Hot Start Polymerase amplifies GC-rich targets more efficiently than other polymerases. Six polymerases shown were used to amplify a 90% GC-containing region of human IGF2R [NM_000876] from HeLa cell cDNA. PCR cycling parameters for KOD Xtreme™ polymerase were: initial denaturation at 94 ºC for 2 min, 30 cycles at 98 ºC for 10 s, and 68 ºC for 9 min. For polymerases from other manufacturers, optimal recommended parameters were used.

OmniPur Agarose PCR Plus

For superior resolution of DNA fragments, especially small fragments (<1000 bp and PCR products), use OmniPur Agarose PCR Plus. OmniPur Products represent a grade of molecular biology reagents that are of the highest quality and deliver consistent performance from lot to lot. Each lot of OmniPur grade reagents is tested for the absence of DNase, RNase, and protease for safe use in tissue and cell culture applications. OmniPur Agarose PCR Plus features average gel strength, standard melting and gelling ranges, and is specifically designed to prevent smearing or high flurorescence backgrounds. Plus, this low electroendosmosis (EEO) agarose offers high electrophoretic mobility for shorter electrophoretic runs.

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