Bst DNA Polymerase, Exonuclease Minus

8,000 U/mL

Includes 10X DNA Polymerase Buffer B (1.2 ml per 2,000 Units)

Store at –20 ºC.

For Research Use Only. Not for use in Diagnostic Procedures.

Technical Specifications

Product Description

Bst DNA Polymerase, Exonuclease Minus, 8,000 units/mL.

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.

Stability

Bst DNA Polymerase, Exonuclease Minus is stable for one year from the date received if stored at –20 ºC.

Recommended Reaction Conditions

8 U Bst DNA Polymerase, Exonuclease Minus; 1X DNA Polymerase Buffer B containing 20 mM Tris-HCl pH 8.8, 10 mM (NH4)²SO⁴, 10 mM KCl, 2 mM MgSO⁴,and 0.1 % Triton X-100.

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 [³³P]dCTP), and 0.1 mg/mL BSA.

Absence of Endonuclease or Nicking Activity

Incubation of 8 U of Bst DNA Polymerase, Exonuclease Minus with 1 µg of pUC19 DNA for 16-18 hours at 37 ºC resulted in no smearing of bands as detected by agarose gel electrophoresis.

Absence of Exonuclease Activity

Incubation of 8 U of Bst DNA Polymerase, Exonuclease Minus with 1 µg of HindIII-cut lambda DNA for 16 hours at 37 ºC and 65 ºC 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/ µLwith radiolabeled DNA substrate for one hour at 37 ºC and 65 ºC, resulting in less than 0.1% release of TCA-soluble counts.

Purity

>90% 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.

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