VeraSeq 2.0 High-Fidelity DNA polymerase is an engineered, ultra-thermostable polymerase designed to maximize the speed, accuracy, and length of DNA synthesis during sequencing template preparation. The result is a novel enzyme that can extend a kilobase of sequence in 15 seconds and with an accuracy 50 times higher than Taq DNA Polymerase.
Source of Protein
A recombinant E. coli strain carrying the engineered VeraSeq 2.0 gene.
20 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
pH 7.4 @ 25°C
5X VeraSeq Buffer II (B7102)
5X VeraSeq GC Buffer (B7130)
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.
|Storage Temperature||-25 to -15°C|
|Specific Activity||100,000 U/mg|
|DS Endonuclease||120 U = no conversion|
|E.coli DNA Contamination||150 U < 10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
QUALITY CONTROL ANALYSIS
Unit Characterization Assay
Specific activity was measured using a 2-fold 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 (OD280) Measurement
The enzyme was analyzed at OD280 using a Nanodrop spectrophotometer standardized with a 2.0 mg/ml BSA sample and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 134,130 and molecular weight of 97,697 Daltons.
SDS-Page (Physical Purity Assessment)
A concentrated sample of enzyme was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample. Comparison between the concentrated and the diluted samples is used to evaluate percent purity.
Double-Stranded Endonuclease Activity
A reaction containing plasmid DNA incubated with enzyme solution for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate samples of enzyme were denatured and screened in a TaqMan qPCR assay for the presence of contaminatingE.coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.