Bst X DNA Polymerase

• OEM by QIAGEN offers bulk manufacturing of Bst X DNA Polymerase in custom formulations
• Thermostable DNA polymerase (Bst)
• No 5'→3' exonuclease activity
• No 3'→5' exonuclease activity
• Exhibits strong strand displacement activity allowing it to perform well in isothermal amplification technologies
• Ask about low glycerol formulations

Bst X DNA Polymerase is a recombinant, thermostable DNA polymerase from Geobacillus that is homologous to the well-known DNA polymerase from Bacillus stearothermophilus (Bst). The Bst X enzyme is similar to Bst polymerase in that it lacks 5'→3' and 3'→5' exonuclease activity and has a potent strand-displacement functionality. 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 MgSO₄ at 1x concentration. Depending on the application, it may be necessary to adjust the MgSO₄ 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

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 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.

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.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.
• Isothermal amplification