Bst DNA Polymerase, Large Fragment

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cloned at neb recombinant unique buffer incubation temp heat inactivation
Catalog #SizeConcentrationPriceQtyAdd to Cart
M0275S1,600 units8,000 units/ml$67.00Add to Cart
M0275L8,000 units8,000 units/ml$268.00Add to Cart
M0275M8,000 units120,000 units/ml$268.00Add to Cart
  
Categories:
Isothermal Amplification & Strand Displacement,
Isothermal Amplification & Strand Displacement
Applications:
Isothermal Amplification,
Isothermal Amplification,
Whole Genome Amplification

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 Pack-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.

Reaction Conditions

1X ThermoPol® Reaction Buffer
Incubate at 65°C

1X ThermoPol® Reaction Buffer:
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1% Triton® X-100
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 7.1 @ 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, including CutSmart?
  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?
  17. Does NEB have a master mix for LAMP or RT-LAMP reactions?

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 Charts

Usage Guidelines & Tips

Troubleshooting Guides

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. Further information regarding NEB product quality can be found here.
  • 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.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

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.