VeraSeq™ ULtra DNA Polymerase

Ultrathermostable uracil-literate polymerase for high-fidelity PCR

S_2962_GEN_generic
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM

VeraSeq ULtra DNA Polymerase (500 U)

Cat. No. / ID:   P7520L

500 U (evaulation pack) of VeraSeq ULtra DNA Polymerase, 5X VeraSeq Buffer II and 5X VeraSeq GC Buffer.
₹39,690.00
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Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application; therefore, it is the sole responsibility of the users of the product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used. This product is licensed from the University of Newcastle upon Tyne under U.S. Pat. No. 8,557,554.
The VeraSeq™ ULtra DNA Polymerase is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM

Features

  • Read and use uracil residue with maximum speed and accuracy
  • Ultra-thermostable polymerase
  • 25x greater fidelity than Taq DNA polymerase
  • Functions in Mg2+ 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

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

Test Units tested Specification
Purity n/a > 95%
Specific activity n/a 100,000 U/mg
Double-stranded endonuclease 120 U No conversion
E. coli DNA contamination 150 U <10 copies

 

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

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.

Reaction setup (for 50 µl)
Component Volume (µl) Final concentration
Sterile H2O Variable n/a
5X VeraSeq™ buffer II or 5X VeraSeq™ GC buffer ULtra 1X
10 mM dNTP mix 1 200 µM each
Primer 1 Variable 0.2 µM
Primer 2 Variable 0.2 µM
DNA template Variable See note 4 below
VeraSeq™ ULtra DNA Polymerase 0.5 1 U

Total reaction volume can be adjusted as needed

Typical cycling conditions
Step Temperature Time Cycles
Initial denaturation 98°C 30 seconds 1
Denaturation
Annealing
Extension
98°C
Varies
72°C
5–10 seconds
10–30 seconds
15–30 seconds/kb
15–35
Final extension 72°C
4°C
5–10 min
Hold
1
Cycling conditions may need to be optimized, depending on the amplicon of interest

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:

Complexity Source example Guideline
Low Plasmid, virus, BAC 1 pg – 10 ng
High Genomic DNA 50–250 ng

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 MgCl2 of 1.5 mM. In cases where additional Mg2+ is required, adjust the final Mg2+ 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 OD280 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

This product is available for molecular biology applications such as:

  • High-fidelity DNA amplification
  • Cloning
  • Synthetic biology

Resources

Quick-Start Protocols (1)
Safety Data Sheets (1)
Certificates of Analysis (1)

FAQ

When should I use 5X VeraSeq GC Buffer?
VeraSeq GC Buffer is recommended for use with difficult or GC-rich amplicons. GC Buffer may also improve yield of some targets. VeraSeq GC Buffer is also recommended for extended room temperature incubation of VeraSeq ULtra DNA polymerase with reaction components prior to PCR cycling. 
FAQ ID - 3923
How is VeraSeq ULtra DNA Polymerase different from the standard recombinant Taq-B DNA Polymerase?
VeraSeq ULtra DNA Polymerase has higher fidelity, speed, and performance compared to Taq-B DNA Polymerase. VeraSeq ULtra DNA Polymerase has an error rate that is 25 times lower than Taq-B DNA Polymerase and can extend 1 kb of sequence per 15 seconds, drastically reducing cycling times. VeraSeq ULtra DNA Polymerase produces blunt end products whereas Taq-B leaves a single-base 3’ overhang. 
FAQ ID - 3921
What is the fidelity/error rate of VeraSeq ULtra DNA Polymerase?
VeraSeq ULtra DNA Polymerase has an error rate of 1.0 x 10-6 in VeraSeq Buffer II and 7.3 x 10-7 in VeraSeq GC Buffer when measured using a LacI-based assay (2).
FAQ ID - 3925
How can yield for targets be increased when using VeraSeq ULtra DNA Polymerase?
Use of a PCR enhancer containing Betaine, DTT, BSA, and DMSO (3) may also be used to improve yield of complex targets (especially targets ≥ 3 kb). Increasing the extension time, the template concentration, or polymerase concentration to 2 U/50 µL may also be helpful. 
FAQ ID - 3926
Do the DNA fragments generated by VeraSeq ULtra DNA Polymerase have a single-base 3´ overhang?
No. VeraSeq ULtra DNA Polymerase generates blunt end products. 
FAQ ID - 3928
What annealing temperature should be used in the cycling conditions?
Set the annealing temperature approximately 3°C higher than the lowest Tm primer for oligos greater than 20 nucleotides. For oligos shorter than 20 nucleotides, set the annealing temperature equal to the Tm of the lowest Tm primer.  When the Tm of the primer pairs is ≥ 72°C, use two-step cycling conditions where the annealing and extension steps are combined. If DMSO is needed in the reaction, a reduction of the annealing temperature is often necessary. 
FAQ ID - 3932
What denaturation temperature should be used in the cycling conditions?
VeraSeq ULtra DNA Polymerase is highly thermostable and it is recommended that denaturation should be performed at 98°C. The initial denaturation time can be extended from 30 seconds up to 3 minutes for difficult templates. For subsequent denaturation cycles, 5-10 seconds at 98°C is sufficient for most targets. 
FAQ ID - 3931
What is the amplification length limit of VeraSeq ULtra DNA Polymerase?
VeraSeq ULtra DNA Polymerase has been demonstrated to amplify up to 5 kb of human genomic DNA and up to 8 kb for lambda DNA.  
FAQ ID - 3924
How can I optimize Mg2+ conditions for a specific amplicon when using VeraSeq ULtra DNA Polymerase and the supplied reaction buffers?
VeraSeq Buffer II and VeraSeq GC buffer result in a final concentration of 1.5 mM Mg2+ in the reaction. Therefore, final Mg2+ reaction concentration may be increased according to user preference with a concentrated solution containing Mg2+.  VeraSeq ULtra DNA Polymerase works under a broad range of Mg2+ concentrations (1.5 – 3.0 mM) but higher Mg2+ can compromise fidelity (1).     
FAQ ID - 3922
Will VeraSeq ULtra DNA Polymerase incorporate dUTP?
Yes.
FAQ ID - 3927
Is VeraSeq ULtra DNA Polymerase available as a hot start enzyme?
No. Currently VeraSeq ULtra DNA Polymerase is not available with a hot start function as a standard product.
FAQ ID - 3929