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  • Bst 2.0 DNA Polymerase

    Description

    Bst 2.0 DNA Polymerase is an in silico designed homologue of Bacillus stearothermophilus DNA Polymerase I, Large Fragment (Bst DNA Polymerase, Large Fragment). Bst 2.0 DNA Polymerase contains 5´→3´ DNA polymerase activity and strong strand displacement activity but lacks 5´→3´ exonuclease activity. Bst 2.0 DNA Polymerase displays improved amplification speed, yield, salt tolerance and thermostability compared to wild-type Bst DNA Polymerase, Large Fragment.

    Product Source

    Bst 2.0 DNA Polymerase is prepared from an E. coli strain that expresses the Bst 2.0 DNA Polymerase protein from an inducible promoter.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Isothermal Amplification Buffer-2010X

    Advantages and Features

    Applications

    • Isothermal DNA amplification
    • Applications requiring strand-displacement DNA synthesis 
    • DNA sequencing through high GC regions
    • Rapid sequencing from nanogram amounts of DNA template

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 25 nmol of dNTP into acid insoluble material in 30 minutes at 65°C.

    Reaction Conditions

    1X Isothermal Amplification Buffer
    Incubate at 65°C

    1X Isothermal Amplification Buffer Pack:
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    50 mM KCl
    2 mM MgSO4
    0.1% Tween® 20
    pH 8.8 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1% Triton® X-100
    pH 7.5 @ 25°C

    Heat Inactivation

    80°C for 20 min

    Unit Assay Conditions

    50 mM KCl, 20 mM Tris- HCl (pH 8.8), 10 mM MgCl2, 30 nM M13mp18 SS DNA, 70 nM M13 sequencing primer (–47) 24 mer, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 100 μM dTTP including [3H]-dTTP and 100 μg/ml BSA.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation there is no detectable phosphatase activity as determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (16 Hour Digestion):

      The product is tested in a reaction containing a RNA substrate.  After incubation for 16 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. Bst 2.0 DNA Polymerase does not exhibit 3´→ 5´ exonuclease activity.
    2. Reaction temperatures above 70°C are not recommended.
    3. Bst 2.0 DNA Polymerase cannot be used for thermal cycle sequencing or PCR.
    4. Specific reaction conditions will vary for different isothermal amplification applications. For best results, use 1X Isothermal Amplification Buffer.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. What is the difference between Bst DNA Polymerase, Large Fragment and Bst 2.0 DNA Polymerase?
    2. Why would I use Bst 2.0 WarmStart DNA Polymerase?
    3. Can Bst DNA 2.0 Polymerase be used in other NEBuffers?
    4. Can Bst 2.0 DNA Polymerase be used to blunt DNA?
    5. Can Bst 2.0 DNA Polymerase be used to fill in 3' overhangs?
    6. Can Bst 2.0 DNA Polymerase be used to remove 5' overhangs?
    7. Can Bst 2.0 DNA Polymerase be heat inactivated?
    8. Are NEB DNA Polymerases supplied with dNTPs?
    9. What are the main causes of reaction failure using Bst 2.0 DNA Polymerase?
    10. Does Bst 2.0 DNA Polymerase have an active 3'→5' proofreading exonuclease?
    11. Can Bst 2.0 DNA Polymerase be used in applications requiring thermal cycling?
    12. Can Bst 2.0 DNA Polymerase initiate at a nick in the DNA?
    13. Can Bst 2.0 DNA Polymerase be used in labeling reactions and partial fill in reactions?
    14. Can Bst 2.0 DNA Polymerase be diluted?
    15. When should Bst 2.0 DNA Polymerase be the enzyme of choice?
    16. Can Bst 2.0 DNA Polymerase be used at temperatures other than 65°C?
    17. Does Bst 2.0 DNA polymerase incorporate dUTP?
    18. Does Bst 2.0 DNA polymerase have reverse transcriptase activity?

    Troubleshooting Guides

    NEB Publications

    • Tanner NA, Evans TC Jr. (2014) Curr Protoc Mol Biol PubMedID: 24510439