本公司生产的Bst DNA Polymerase, Large Fragment 即嗜热脂肪芽孢杆菌(Bacillus stearothermophilus, Bst) DNA Polymerase I 大片段，具有5'→3' DNA 聚合酶活性，不具有 3'→5'和 5'→3'的核酸外切酶活性。Bst DNA Polymerase, Large Fragment 具有很强的链置换(stranddisplacement)能力，可应用于核酸的等温扩增反应，如环介导的等温扩增(Loop-mediated isothermal amplification, LAMP)和滚环扩增(Rolling-circle amplification, RCA)等。Bst DNA Polymerase, Large Fragment 介导的等温扩增温度一般在 50-68℃之间，通常为 65℃。与Bst DNA Polymerase 2.0 相比，本产品在进行等温扩增时不具有将 dUTP 掺入合成的 DNA 链的能力。Bst DNA Polymerase 具有 5'→3'的核酸外切酶活性，而 Bst DNA Polymerase, Large Fragment 通过删除突变而缺失了 5'→3'核酸外切酶活性。
组分和说明
| 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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产品应用
环介导等温扩增 LAMP、解旋酶等温基因扩增(HDA)等 DNA 等温扩增，多重置换扩增(MDA)，全基因组扩增(WGA)，高 GC 含量 DNA 的测序，纳克级 DNA 模板的快速测序，建库测序等。
产品优势
酶活性好，在进行等温扩增时不具有将 dUTP 掺入合成的 DNA 链的能力。强大的链置换能力、高灵敏度和高特异性、高保真性和热稳定性。
使用说明
1.以 LAMP 等温扩增为例，参考下表设置反应体系。
| 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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注 1：在配制反应体系完成后，可每 25μl 体系的反应管管盖加适量高浓度的 SYBR Green I 1μl，待等温扩增反应结束后，8000g 离心 1min，反应体系变荧光绿为阳性，保持无色或棕色为阴性。也可以无需添加指示剂，待反应程序结束后，可见反应液明显变浑浊为阳性，反应液保持透明为阴性。
注 2：如需优化反应，可调整 Mg²⁺浓度(4-10mM)，酶量(0.04-0.32U/μl)或改变反应温度(50-68℃)。
注 3：如果通过琼脂糖凝胶电泳或其它需要打开 LAMP 反应容器的方法进行分析，请设置辅助分析区域和设备，以避免污染。
注 4：由于反应比较迅速，为了确保实验的重现性，建议模板 DNA 最后加入。
注 5：强烈建议设置未加模板的阴性对照，以确保扩增的特异性。
注 6：为了防止在配置试剂时发生污染，请务必在超净工作台内进行操作。
注 7：试剂及模板 DNA 的配置操作尽量需要和 PCR 产物的电泳等分析在不同的区域进行，以免发生污染。
2. 反应程序：65℃ 60min。
3. 灭活：80℃ 20min。
4. 如实验需要，可以用1.5%的琼脂糖凝胶进行电泳分析，电泳图显示扩增产物为梯度条带为阳性，无梯度条带为阴性。
注意事项：
（1）Bst DNA Polymerase, Large Fragment 不具有 3'→5'核酸外切酶活性。
（2）建议等温扩增反应温度不能高于 70℃，否则会导致酶失活。
（3）Bst DNA Polymerase，Large Fragment 不能用于热循环测序和 PCR。
（4）每次等温扩增实验注意设置无模板 DNA 作为阴性对照。
（5）本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
（6）为了您的安全和健康，请穿实验服并戴一次性手套操作。
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.
ZJ26F0838405
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