The Bst DNA Polymerase, Large Fragment produced by our company is the large fragment of Bacillus stearothermophilus (Bst) DNA Polymerase I. It possesses 5'→3' DNA polymerase activity, while lacking both 3'→5' and 5'→3' exonuclease activities.Bst DNA Polymerase, Large Fragment exhibits potent strand displacement capacity, making it applicable to nucleic acid isothermal amplification reactions, including Loop-mediated isothermal amplification (LAMP), Rolling-circle amplification (RCA), and other related assays. Isothermal amplification reactions mediated by Bst DNA Polymerase, Large Fragment are generally performed at 50–68 °C, with 65 °C as the typical working temperature.Compared with Bst DNA Polymerase 2.0, this product cannot incorporate dUTP into newly synthesized DNA strands during isothermal amplification. Full-length Bst DNA Polymerase retains intrinsic 5'→3' exonuclease activity, whereas Bst DNA Polymerase, Large Fragment loses this 5'→3' exonuclease activity via deletion mutation.
Components List
| B744266 | Component | 800U | 5×800U | Storage | | B744266A | Bst DNA Polymerase, Large Fragment (8U/μL) | 100µl | 5×100µl | -20℃. Avoid freeze/thaw cycle. | | B744266B | 10× Bst Reaction Buffer
| 300µl | 5×300µl | -20℃. Avoid freeze/thaw cycle. | | B744266C | 100mM MgSO₄ | 200µl | 5×200µl | -20℃. Avoid freeze/thaw cycle. |
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Product Applications
DNA isothermal amplification techniques including Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Multiple Displacement Amplification (MDA), Whole Genome Amplification (WGA); sequencing of high-GC-content DNA, rapid sequencing of nanogram-level DNA templates, library construction and sequencing, etc.
Product Advantages
High enzymatic activity; incapable of incorporating dUTP into newly synthesized DNA strands during isothermal amplification. It features robust strand displacement capacity, high sensitivity and specificity, excellent fidelity and thermal stability.
Instructions for Use
1. Taking LAMP isothermal amplification as an example, prepare the reaction system according to the reference table below.
| Reagent | Volume | Final concentration | | Nuclease-Free | Water (13.5-x)μl | - | | 10× Bst Reaction Buffer | 2.5μl | 1× | | MgSO₄ (100mM) | 1.5μl 6mM | (8mM total) | | dNTP (10mM each) | 3.5μl | 1.4mM each | | FIP/BIP Primers (25×, 40μM) | 1μl | 1.6μM | | F3/B3 Primers (25×, 5μM) | 1μl | 0.2μM | | LoopF/B Primers (25×, 10μM) | 1μl | 0.4μM | | Template | xμl | >10 copies or more | | Bst DNA Polymerase, Large Fragment (8U/μl) | 1μl | 320U/ml | | Total volume | 25μl | - |
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Note 1: After preparing the reaction system, add 1 μL of high-concentration SYBR Green I to the cap of each reaction tube with a 25 μL reaction system. After the isothermal amplification reaction, centrifuge at 8000 g for 1 min. A fluorescent green reaction mixture indicates a positive result, while a colorless or brown mixture indicates a negative result. Alternatively, no indicator is required; after the reaction completes, obvious turbidity in the reaction solution represents a positive result, and clear solution represents a negative result.
Note 2: For reaction optimization, adjust the Mg²⁺ concentration (4–10 mM), enzyme dosage (0.04–0.32 U/μL), or reaction temperature (50–68 °C).
Note 3: If analysis is performed via agarose gel electrophoresis or other methods that require opening LAMP reaction vessels, prepare separate dedicated working areas and equipment to prevent cross-contamination.
Note 4: The amplification proceeds rapidly. To guarantee experimental reproducibility, add template DNA last.Note 5: A no-template negative control is strongly recommended to verify amplification specificity.Note 6: All reagent preparation steps must be performed in a biosafety cabinet to avoid contamination during setup.Note 7: Prepare reagents and template DNA in an area physically separated from post-reaction processes such as PCR product electrophoresis to prevent contamination.
2. Reaction protocol: Incubate at 65 °C for 60 min.
3. Enzyme inactivation: Heat at 80 °C for 20 min.
4. If further analysis is required, perform electrophoresis using a 1.5% agarose gel. A ladder-like band pattern of amplification products on the gel indicates a positive result; the absence of ladder bands indicates a negative result.
Precautions
(1) Bst DNA Polymerase, Large Fragment lacks 3'→5' exonuclease activity.
(2) The reaction temperature for isothermal amplification is recommended not to exceed 70 °C; otherwise, the enzyme will be inactivated.
(3) Bst DNA Polymerase, Large Fragment is not suitable for thermal cycle sequencing or standard PCR.
(4) A no-template negative control shall be included in every isothermal amplification experiment.
(5) This product is for scientific research use by professional personnel only. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical production, nor stored in residential premises.
(6) Wear a lab coat and disposable gloves during operation for your personal safety and health.
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