Description
Bst X DNA Polymerase is a recombinant, thermostable DNA polymerase from Geobacillus that is homologous to the well-known DNA polymerase from B acillus stearothermophilus (Bst). The Bst X enzyme is similar to Bst polymerase in that it lacks 5'→3' and 3'→5' exonuclease activity and exhibits strong strand displacement activity allowing it to perform well in LAMP isothermal amplification technologies. Relative to Bst polymerase, however, Bst X displays improved amplification speed, salt tolerance, and reaction sensitivity. Bst X DNA Polymerase is supplied in 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, <0.1% Triton X-100, 50% glycerol and 10 mM Tris-HCl; pH 7.5 at 25°C. Bst X DNA Polymerase is provided with 10x Xcelerator Reaction Buffer (B7390) and 100 mM Magnesium Sulfate Solution. 10x Bst X Xcelerator Reaction Buffer (B7390) contains 2 mM MgSO 4 at 1x concentration. Depending on the application, it may be necessary to adjust the MgSO 4 from a final concentration of 2 mM to a 10 mM. Ask about low glycerol formulations
Properties Optimal reaction temperature from 60-70°C. Heat inactivated at 80°C after 10 min incubation. High tolerance to the non-ionic detergents Tween-20, Tween-80, Triton X-100, and NP-40 (up to 5% final concentration). Active in salt from 40 to 125 mM. • Alternate temperature Approximately 15% activity at 37°C. Approximately 5% activity at 25°C Storage temperature: –25°C to –15°C Molecular weight: 66.5 kDa Test Specification Purity >99% Specific activity 400,000 U/mg Single-stranded exonuclease 4000 U; <5.0% released Double-stranded exonuclease 4000 U; <1.0% released Double-stranded endonuclease 4000 U; no conversion E. coli DNA contamination 4000 U; < 10 copies
Source of recombinant enzyme protein The protein is produced by a recombinant E. coli strain carrying the Bst X DNA Polymerase (exo-) polymerase gene. Unit definition: One unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material in 30 minutes at 65°C.
Quality control analysis Unit activity is measured using a twofold serial dilution method. Dilutions of enzyme batch were made in 1x reaction buffer and added to 50 μl reactions containing calf thymus DNA, 1x PCR Buffer II, 3H-dTTP and 100 μM dNTPs. Reactions were incubated 10 minutes at 65°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26). 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. Single-stranded exonuclease is 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 activity is 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 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.
