Description
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.
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
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.
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 H 2 O 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 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.
