Bst DNA Polymerase, Exonuclease Minus, is a recombinant form of the 67 kDa Bacillus stearothermophilus DNA Polymerase protein (large fragment). The enzyme has 5’-3’ polymerase activity and strand displacement activity, but it lacks 3’-5’ exonuclease activity. It also has reverse transcription activity.
Lucigen's Bst DNA Polymerase, Exonuclease Minus, has higher strand displacement activity than that of other suppliers (Figure 1). The enzyme can be used in nucleic acid amplification methods* such as isothermal amplification, whole genome amplification (WGA), and multiple displacement amplification (MDA). It also can be used in Next Generation sequencing.
This enzyme has optimal activity at 65°C. It is suitable for sequencing DNA with high GC content and secondary structures. It is available in concentrations of 8,000 U/ml or 50,000 U/ml.
Bst DNA Polymerase, Exonuclease Minus, is supplied with 10X DNA Polymerase Buffer B, composed of 200 mM Tris-HCl pH 8.8, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, and 1.0 % Triton X-100.
Applications
- Strand displacement amplification*
- DNA sequencing through high GC regions (1,2)
- Rapid sequencing from nanogram amounts of DNA template (3)
Tips for use:
- Requires 0.1% Triton X-100 for long term storage.
- Reaction temperatures above 70°C are not recommended.
- Bst DNA Polymerase cannot be used for thermal cycle sequencing.
Heat Inactivation: 80ºC for 20 min.
*Note:
Some uses for this product may require licenses. Lucigen does not encourage or support the unauthorized or unlicensed use of patented nucleic acid amplification processes for isothermal amplification, whole genome amplification (WGA), multiple displacement amplification (MDA), and Next Generation sequencing. It is the sole responsibility of the buyer to ensure that use of the product does not infringe the patent rights of third parties. If the purchaser is not willing to accept these use limitations, Lucigen Corporation is willing to accept return of the product for a full refund.
Storage Buffer: 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, and 50% Glycerol.
Purity: >99% pure by SDS-PAGE. No detectable DNA contamination. 10 µl of enzyme at 8 U/µl of the sample was tested for E. coli genomic DNA contamination by PCR amplifying with the E. coli 16S ribosomal primers.
Activity Determination: One unit catalyzes the incorporation of 10 nmol of dNTP into acid-insoluble material in 30 minutes at 65°C in 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1 % Triton X-100, 30 nM M13mp18 ssDNA, 70 nM M13 sequencing primer(-47) 24 mer 200 µM dGTP, dATP, dTTP, dCTP (a mix of unlabeled and and 33P]dCTP), and 0.1 mg/ml BSA.
Absence of Endonuclease or Nicking Activity: Incubation of 8 U and 50 U of Bst DNA Polymerase, Exonuclease Minus, with 1 µg of supercoiled pBR322 DNA for 16 hours at 37° and 65°C resulted in no detectable conversion to relaxed or linear forms by agarose gel electrophoresis.
Absence of Exonuclease Activity: Incubation of 8 U and 50 U of Bst DNA Polymerase, Exonuclease Minus, with 1 µg of HindIII-cut lambda DNA for 16 hours at 37° and 65°C resulted in no smearing of bands on agarose gels. Single stranded and double stranded exonuclease activities were tested by incubating 10 µl of enzyme at 8 U/µl with radiolabeled DNA substrate for one hour at 37° and 65°C, resulting in less than 0.1% release of TCA-soluble counts.
