Description
Taq-B DNA Polymerase is a thermally stable, processive, 5′→3′ DNA polymerase from the thermophilic organism Thermus aquaticus YT-1. The 94 kDa protein possesses an inherent 5′→3′ exonuclease activity and lacks a 3′→5′ proofreading function. Taq excels at amplifying shorter (<5 kb) sequences from low complexity template sources and produces robust yields with little or no optimization of reaction conditions. The enzyme is supplied in 20mM Tris-HCl, 100mM NaCl, 1mM DTT, 0.1mM EDTA, Stabilizer, 50% glycerol pH 7.5 at 25⁰C. It is supplied with a 10X PCR Buffer l (B7030) containing: 100mM Tris-HCl, 500mM KCl, 15mM MgCl 2 pH 8.3 at 25⁰C.
Assay Specification Purity >99% Specific activity 74,625 U/mg Single-stranded exonuclease 50 U; functional Double-stranded exonuclease 50 U; functional Double-stranded endonuclease 50 U; no conversion E. coli DNA contamination 50 U; <10 copies
The enzyme catalyzes 5'→3' synthesis of DNA, has no detectable 3'→5' exonuclease (proofreading) activity and possesses 5'→3' exonuclease activity.
Instructions for using Taq-B DNA Polymerase are provided in the corresponding kit protocol in the resources below. Quality Control Unit activity was measured using a two-fold serial dilution method. Dilutions of the enzyme were made in 1X reaction buffer and added to 50 μL reactions containing calf thymus DNA, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0mM MgCl 2, 1 mM DTT, 3H-dTTP and 100 μM dNTPs. Reactions were incubated for 10 minutes at 75°C, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26). Protein concentration was determined by OD 280 absorbance. Physical purity was 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 protein of interest band in the diluted sample. Single-stranded exonuclease was determined in a 50 μL reaction containing a radiolabeled single-stranded DNA substrate and 10 μL of enzyme solution incubated for 4 hours at 37°C. Double-stranded exonuclease was determined in a 50 μL reaction containing a radiolabeled double-stranded DNA substrate and 10 μL of enzyme solution incubated for 4 hours at 37°C. Double-stranded endonuclease was 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 was evaluated using 5 μL replicate samples of enzyme solution 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.
