Bst X DNA Polymerase

Bst X DNA Polymerase is a thermostable DNA polymerase from Geobacillus, homologous to the DNA polymerase from Bacillus stearothermophilus (Bst). Bst X lacks 5’→3′ and 3’→5′ exonuclease activity and exhibits a stronger strand-displacement activity than Manta 1.0 DNA Polymerase that may allow it to perform better in isothermal amplification technologies.

Usage Notes:

• 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, TX-100, and NP-40 (up to 5% final concentration).
• Active in salt from 40 to 125 mM.

Buffer Description
10x Bst X Xcelerator Reaction Buffer, contains 2 mM MgSO₄ at 1X concentration. Depending on the application, it may be necessary to adjust the MgSO₄ from 2-10 mM final concentration.

Source of Protein
A recombinant E.coli strain carrying the Bst X DNA Polymerase (exo-) polymerase gene.

Unit Definition
1 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

Molecular weight
66,494 Daltons

* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.

QUALITY CONTROL ANALYSIS

Unit Activity is measured using a 2-fold 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, plunged 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 protein of interest band in the diluted sample.

Single-Stranded Exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10µL of enzyme solution incubated for 4 hours at 37°C.

Double-Stranded Exonuclease 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 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 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.

Supplied in
50 mM KCl
1mM DTT
0.1mM EDTA
0.1% Triton X-100
50% glycerol
10mM Tris-HCl
pH 7.5 @ 25°C.

LEGAL DISCLAIMER

Patents
Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application; therefore it is the sole responsibility of users of this product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used.