Qiagen, P7520L, VeraSeq ULtra DNA Polymerase (500 U)

500 U of VeraSeq ULtra DNA Polymerase, 5X VeraSeq Buffer II and 5X VeraSeq GC Buffer.

Features

- Read and use uracil residue with maximum speed and accuracy
- Ultra-thermostable polymerase
- 25x greater fidelity than Taq DNA polymerase
- Functions in Mg 2+ concentrations 1.5 to 3.0 mM
- Strong proofreading function (3'-5' exonuclease)
- OEM by QIAGEN offers bulk manufacturing of VeraSeq ULtra DNA Polymerase in custom formulations, including low-glycerol or hot-start formulations

Product Details

VeraSeq™ ULtra DNA Polymerase is an engineered, ultra-thermostable, uracil-literate polymerase designed to maximize the speed, accuracy, and length of DNA synthesis during sequencing template preparation. The result is a novel enzyme that can read through uracil, extend a kilobase of sequence in 15 seconds and has an accuracy 25 times higher than Taq DNA Polymerase.

Supplied in: 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, stabilizer and 50% glycerol; pH 7.4 at 25°C.

Supplied with: 5X VeraSeq™ Buffer II (B7102L) and 5X VeraSeq™ GC Buffer (B7130L).

You can ask us about low-glycerol or hot-start formulations.

Performance

Test: Units tested
Purity: n/a
Specific activity: n/a
Double-stranded endonuclease: 120 U
E. coli DNA contamination: 150 U

Polymerase properties Extension rate : 15 seconds per kilobase at 72˚C Proofreading (3'-5' exo) : Yes, strong Nick-translation (5'-3' exo) : No Fidelity : > 25X higher than Taq DNA Polymerase Strand displacement : No Thermostability : Highly thermostable Able to extend an RNA primer : No Extends from a nick : No Generate blunt end products : Yes Uracil read through : Yes Storage temperature : –25°C to –15°C

Principle

Source of recombinant enzyme protein The protein is produced by a recombinant E. coli strain carrying the engineered VeraSeq™ ULtra gene.

Unit definition: One unit is defined as the amount of enzyme required to incorporate 10 nmoles of dNTPs into acid-insoluble form at 74°C in 30 minutes.

Procedure

Component: Volume (µl)
Sterile H 2 O: Variable
5X VeraSeq™ buffer II or 5X VeraSeq™ GC buffer: ULtra
10 mM dNTP mix: 1
Primer 1: Variable
Primer 2: Variable
DNA template: Variable
VeraSeq™ ULtra DNA Polymerase: 0.5

Step: Temperature
Initial denaturation: 98°C
Denaturation Annealing Extension: 98°C Varies 72°C
Final extension: 72°C 4°C
Cycling conditions may need to be optimized, depending on the amplicon of interest

Complexity: Source example
Low: Plasmid, virus, BAC
High: Genomic DNA

Protocol General precautions should be taken when setting up PCR, including setting up the reaction on ice, adding the polymerase last, gentle pipetting, thorough mixing and a quick centrifugation. The following procedure can be used as a guideline. Reactions may need to be optimized individually.

Total reaction volume can be adjusted as needed

Usage notes:

1. 5X VeraSeq™ Buffer II should be used as the default buffer for high-fidelity amplification. For GC-rich and difficult templates, use 5X VeraSeq™ GC Buffer 2. VeraSeq™ 2.0 ULtra DNA Polymerase can be used in PCR amplification to generate deoxyuridine-containing products by including dUTP in the reaction. If DNA template contains uracil or dUTP needs to be incorporated, use VeraSeq Ultra (P7520L) 3. A final concentration of 0.2 µM is recommended for each primer, but it can be varied in the range of 0.2–1 µM 4. Recommended template quantities:

5. One unit is usually sufficient for amplifying most targets. For long targets (>1kb), difficult templates or to increase yield, it may be necessary to add up to 2 units of enzyme.

6. Both 5X VeraSeq™ Buffer II and GC buffer are formulated to provide a final 1X concentration of MgCl 2 of 1.5 mM. In cases where additional Mg 2+ is required, adjust the final Mg 2+ concentration in 0.2 mM steps.

7. For GC rich templates, DMSO may be used to reduce the secondary structure of complex templates. DMSO is generally used at a 3% final concentration (v/v). If additional optimization is required, adjust the concentration in 1–2% increments (2–9% in final reaction). The primer annealing temperature should be lowered to account for the presence of the solvent.

8. VeraSeq™ ULtra DNA Polymerase is also compatible with other PCR-enhancing additives, such as BSA and betaine.

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 µl reactions containing activated calf thymus DNA; 25 mM TAPS (tris-[hydroxymethyl]-methyl-amino-propanesulfonic acid, sodium salt) pH 9.3 at 25°C; 50 mM KCl; 2 mM MgCl2; 1 mM β-mercaptoethanol; 200 µM each dATP, dGTP, dTTP; and 100 µM [3H]-dCTP (0.075 Ci/mmole). Reaction vessels were mixed and incubated at 74°C for 10 minutes.

Protein concentration is determined by OD 280 absorbance.

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli 16S rDNA contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

Applications

- High-fidelity DNA amplification
- Cloning
- Synthetic biology

This product is available for molecular biology applications such as: