VeraSeq 2.0 High-Fidelity DNA Polymerase

OEM by QIAGEN offers bulk manufacturing of VeraSeq 2.0 High-Fidelity DNA Polymerase in custom formulations.

S_1143_6_OEM_Generic_Product
Let’s talk custom
Need this product modified? Contact us to get started with tailored OEM solutions.
VeraSeq 2.0 High-Fidelity DNA Polymerase (500 U/ml)

Cat. No. / ID: P7511L

500 U (evaluation pack) of VeraSeq 2.0 High-Fidelity DNA Polymerase (2000 U/ml) with 5X VeraSeq Buffer II and 5X VeraSeq GC Buffer.
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 Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424,7,541,170,7,560,260 and corresponding patents in other countries for use solely in DNA sequencing, DNA micro-array, and conventional PCR applications, including pre-amplification steps that are required for such applications, in the life science research and in-vitro diagnostics fields but not real-time PCR or digital PCR. The VeraSeq 2.0 High-Fidelity DNA Polymerase is intended for molecular biology applications This product is not intended for the diagnosis, prevention or treatment of a disease.
Let’s talk custom
Need this product modified? Contact us to get started with tailored OEM solutions.

Features

  • Ultra-thermostable polymerase
  • Extends 1 kb in 15 seconds
  • 50x greater fidelity than Taq DNA polymerase
  • Functions in Mg2+ concentrations 1.5–3.0 mM
  • Strong proofreading (3'-5' exonuclease)

Product Details

VeraSeq 2.0 High-Fidelity DNA Polymerase is an engineered, ultra-thermostable polymerase that delivers industry-leading speed, fidelity and robustness to PCR amplification. The novel enzyme that can extend a kilobase of sequence in 15 seconds and with an accuracy 50 times higher than Taq DNA Polymerase.

The enzyme is 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.

VeraSeq 2.0 High-Fidelity DNA Polymerase is supplied with 5x VeraSeq Buffer II (B7102) and 5x VeraSeq GC Buffer (B7130).

Ask about low glycerol or hot-start formulations.

Performance

Polymerase properties
Extension rate: 15 seconds per kb at 72˚C
Proofreading (3'-5' exo): Yes, strong
Nick-translation (5'-3' exo): No

Fidelity: >50x 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: No

  • Storage temperature: –25°C to –15°C
Test Units tested Specification
SDS 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 2.0 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 a PCR, including setting up the reaction on ice, adding 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  
5x VeraSeq Buffer II or 5x VeraSeq GC Buffer 10 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
VeraSeq 2.0 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 mininutes
hold
1
Cycling conditions may need to be optimized, depending on the amplicon of interest

Notes:

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. VeraSeq 2.0 High-Fidelity DNA Polymerase stalls on uracil residues in the template strand and prevents further extension. Therefore, dUTP should not be used in the reaction. If DNA templates contains uracil or dUTP needs to be incorporated, use VeraSeq ULtra (P7520L). 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.

Recommended template quantities:

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

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. 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.

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. VeraSeq 2.0 High-Fidelity DNA Polymerase is also compatible with other PCR-enhancing additives, such as BSA and betaine.

Quality control analysis

Specific 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 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 OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

  • High-fidelity amplification
  • Long amplification
  • Cloning
  • Synthetic biology

Resources

FAQ

Will VeraSeq 2.0 DNA Polymerase incorporate dUTP?
No.  Please see VeraSeq Ultra DNA Polymerase (P7520L).
FAQ ID - 3915
What is the amplification length limit of VeraSeq 2.0 DNA Polymerase?
VeraSeq 2.0 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 - 3912
What denaturation temperature should be used in cycling conditions?
VeraSeq 2.0 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 - 3918
How is VeraSeq 2.0 DNA Polymerase different from the standard recombinant Taq-B DNA Polymerase?
VeraSeq 2.0 DNA Polymerase has higher fidelity, speed, and performance compared to Taq-B DNA Polymerase. VeraSeq 2.0 DNA Polymerase has an error rate that is 50 times lower than Taq-B DNA Polymerase and can extend 1 kb of sequence per 15 seconds, drastically reducing cycling times.

VeraSeq 2.0 DNA Polymerase produces blunt end products whereas Taq-B leaves a single-base 3’ overhang.
FAQ ID - 3908
What is the amplification length limit of VeraSeq 2.0 DNA Polymerase?
VeraSeq 2.0 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 - 3911
Is VeraSeq 2.0 DNA Polymerase available as a hot start enzyme?
Yes.  Please see Hot Start VeraSeq 2.0 P7690-4000.
FAQ ID - 3916
What is the stability of VeraSeq 2.0 DNA Polymerase at room temperature?
VeraSeq 2.0 DNA Polymerase retained greater than 80% activity after incubation in product storage buffer at room temperature for 45 days.
FAQ ID - 3920
How can yield for targets be increased when using VeraSeq 2.0 DNA Polymerase?
Increase the polymerase concentration to 2 U/50µL for targets ≥ 1 kb.  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 and/or the template concentration may also be helpful. 
FAQ ID - 3914
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 2.0 DNA polymerase with reaction components prior to PCR cycling. 
FAQ ID - 3910
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 - 3919
Why is VeraSeq 2.0 DNA Polymerase sometimes cloudy upon removing from -20°C storage?
VeraSeq 2.0 DNA Polymerase solution may appear cloudy immediately upon removing from –20°C storage due to the presence of a stabilizer in the storage buffer.
FAQ ID - 3913
How long can reaction components incubate with VeraSeq 2.0 DNA Polymerase at room temperature prior to PCR cycling?
Successful amplification with high specificity and yield has been achieved after incubating all components for 24 hours prior to PCR cycling. For maximum success, use VeraSeq GC Buffer.  
FAQ ID - 3917
How can I optimize Mg2+ conditions for a specific amplicon when using VeraSeq 2.0 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 2.0 DNA Polymerase works under a broad range of Mg2+ concentrations (1.5 – 3.0 mM) but higher Mg2+ can compromise fidelity (1).     
FAQ ID - 3909