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  • Bst DNA Polymerase, Large Fragment

    Description






    Bst DNA Polymerase, Large Fragment is the portion of the Bacillus stearothermophilus DNA Polymerase protein that contains the 5´ → 3´ polymerase activity, but lacks 5´ →3´ exonuclease activity.

    Highlights

    • Isolated from a recombinant source
    • Sequencing through problematic secondary structures
    • Supplied with 10X Reaction Buffer

    Product Source

    Bst Polymerase, Large Fragment is prepared from an E. coli strain containing a genetic fusion of the Bacillus stearothermophilus DNA Polymerase gene, lacking the 5´ → 3´ exonuclease domain, and the gene coding for E. coli maltose binding protein (MBP). The fusion protein is purified to near homogeneity and the MBP portion of the fusion is cleaved off in vitro. The remaining polymerase is purified free of MBP (1).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ThermoPol® Reaction Buffer-2010X
    Magnesium Sulfate (MgSO4) Solution-20100 mM

    Advantages and Features

    Applications

    • Isothermal amplification (LAMP)
    • DNA sequencing through high GC regions (2,3)
    • Rapid Sequencing from nanogram amounts of DNA template (4)

    Properties and Usage

    Unit Definition

    One unit is defined at the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65°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 7.4 @ 25°C

    Heat Inactivation

    80°C for 20 min

    Molecular Weight

    Theoretical: 67000 daltons

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    No

    Strand Displacement

    ++++

    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.

    Notes

    1. Bst DNA Polymerase does not exhibit 3´→ 5´ exonuclease activity.
    2. 100 µg/ml BSA or 0.1%Triton X-100 is required for long term storage.
    3. Reaction temperatures above 70°C are not recommended.
    4. Bst DNA Polymerase, Large Fragment cannot be used for thermal cycle sequencing or PCR.

    References

    1. Kong, H., Aliotta, J. and Pelletier, J.J. New England Biolabs. Unpublished observation
    2. Griffin, H. and Griffin, A. (1994). PCR Technology. 228-229.
    3. McClary, J. et al. (1991). J. DNA Sequencing and Mapping. 1, 173-180.
    4. Mead, D.A. et al. (1991). Biotechniques. 11, 76-87.
    5. Notomi et al. (2000). Nucleic Acids Res. 28(12), E63.
    6. Hsieh et al. (2014). Chem. Commun. 50, 3747-3749.
    7. Tanner and Evans (2014). Curr. Prot. Mol. Biol. 105, 15.14.

    FAQs

    1. Can Bst DNA Polymerase be used in other NEBuffers?
    2. Can Bst DNA Polymerase be used to blunt DNA?
    3. Can Bst DNA Polymerase be used to fill in 3' overhangs?
    4. Can Bst DNA Polymerase be used to remove 5' overhangs?
    5. Can Bst DNA Polymerase be heat inactivated?
    6. Are NEB DNA Polymerases supplied with dNTPs?
    7. What are the main causes of reaction failure using Bst DNA Polymerase?
    8. Does Bst DNA Polymerase have an active 3'→5' proofreading exonuclease?
    9. Can Bst DNA Polymerase be used for thermal cycle sequencing?
    10. Can Bst DNA Polymerase initiate at a nick in the DNA?
    11. Can Bst DNA Polymerase be used in labeling reactions and partial fill in reactions?
    12. Can Bst DNA Polymerase be diluted?
    13. When should Bst DNA Polymerase be the enzyme of choice?
    14. Can Bst DNA Polymerase be used at temperatures other than 65°C?
    15. Does Bst DNA Polymerase, Large Fragment incorporate dUTP?
    16. Does Bst DNA polymerase have reverse transcriptase activity?

    Protocols

    1. Typical LAMP Protocol (M0275)

    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.

    Selection Tools

    Troubleshooting Guides

    Interactive Tools

    Citations

    • Trisadee Khamlor, Petai Pongpiachan, Rangsun Parnpai, Kanchana Punyawai, Siwat Sangsritavong, Nipa Chokesajjawatee (2015). Bovine embryo sex determination by multiplex loop-mediated isothermal amplification. Theriogenology. 83, 891-6. PubMedID: 25542460, DOI: 10.1016/j.theriogenology.2014.11.025
    • DoKyung Lee, Eun Jin Kim, Paul E Kilgore, Soon Ae Kim, Hideyuki Takahashi, Makoto Ohnishi, Dang Duc Anh, Bai Qing Dong, Jung Soo Kim, Jun Tomono, Shigehiko Miyamoto, Tsugunori Notomi, Dong Wook Kim, Mitsuko Seki (2015). Clinical Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Neisseria meningitidis in Cerebrospinal Fluid. PLoS One. 10, e0122922. PubMedID: 25853422, DOI: 10.1371/journal.pone.0122922
    • Mohammad Reza Allahyar Torkaman, Kazunari Kamachi, Vajihe Sadat Nikbin, Masoumeh Nakhost Lotfi, Fereshteh Shahcheraghi (2015). Comparison of loop-mediated isothermal amplification and real-time PCR for detecting Bordetella pertussis. J Med Microbiol. 64, 463-5. PubMedID: 25596118, DOI: 10.1099/jmm.0.000021
    • Aongart Mahittikorn, Hirotake Mori, Supaluk Popruk, Amonrattana Roobthaisong, Chantira Sutthikornchai, Khuanchai Koompapong, Sukhontha Siri, Yaowalark Sukthana, Duangporn Nacapunchai (2015). Development of a Rapid, Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP). PLoS One. 10, e0120997. PubMedID: 25822175, DOI: 10.1371/journal.pone.0120997
    • Manabu Nemoto, Yoshinori Morita, Hidekazu Niwa, Hiroshi Bannai, Koji Tsujimura, Takashi Yamanaka, Takashi Kondo (2015). Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification. J Virol Methods. 215-216, 13-6. PubMedID: 25682750, DOI: 10.1016/j.jviromet.2015.02.001

    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.
    • Non-Specific DNase Activity (16 hour, DNA):
      The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.
    • 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.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    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.