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Bst 8.0 DNA Polymerase
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概述
本公司生产的 Bst 8.0 DNA Polymerase 是一种 Bst DNA Polymerase, large fragment 的同源蛋白，即嗜热脂肪芽孢杆菌(Bacillus stearothermophilus, Bst) DNA 聚合酶大片段的同源蛋白，和 Bst DNA Polymerase 大片段相比，具有更强的 5'→3' DNA 聚合酶活性、更强的链置换(Strand displacement)能力、dUTP 耐受性、耐盐性以及非离子去垢剂耐受性。Bst 8.0 DNA Polymerase 不具有 5'→3'和 3'→5'的核酸外切酶活性，可应用于环介导的等温扩增(Loop-mediated isothermal amplification, LAMP)、交叉引物扩增技术(Crossing priming amplification, CPA)、滚环扩增(Rolling-circle amplification, RCA)和基于滚环扩增的等温扩增反应等。Bst 8.0 DNA Polymerase 介导的等温扩增温度一般在 50-72 °C 之间，通常为 65 °C。最佳温度与所使用的引物和扩增的产物有关，需要通过实验进行优化。
| 来源（Source） | 大肠杆菌重组表达 |
| 外观（Appearance） | 无菌液体 |
| 保存液（Storage Buffer） | 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 @ 25 °C)。 |
| 酶浓度（Enzyme Concentration） | 40 U/μL |
| 纯度（Purity） | 无 DNA 内切酶和外切酶活性。 |
| 活性定义（Activity Definition） | One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65 °C。 |
组分和说明
| 组分 | B744265-8KU | B744265-40KU |
|---|---|---|
| Bst 8.0 DNA Polymerase (40 U/μL) | 200 μL | 1 mL |
| 10 × Bst 8.0 Reaction Buffer | 600 μL | 3 mL |
| 100 mM MgSO₄ | 400 μL | 2 mL |
产品应用
环介导等温扩增(LAMP)、逆转录环介导等温扩增(RT-LAMP) (反应温度可达 72 °C)、交叉引物扩增技术(CPA)、滚环扩增(RCA)和解旋酶等温扩增(HDA)等 DNA 等温扩增，多重置换扩增(MDA)，链置换 DNA 合成，全基因组扩增(WGA)，高 GC 含量的 DNA 的测序，纳克级 DNA 模板的快速测序，建库测序等。
产品优势
本产品不仅具有相当的 DNA 聚合酶活性、链置换能力、dUTP 耐受性和耐高温特性(可耐受 72 °C)；并具有更好的逆转录酶活性和更强的 dUTP 耐受性，可应用于逆转录环介导的等温扩增(Reverse Transcription Loop-mediated isothermal amplification, RT-LAMP)。
使用说明
1. 引物设计。
环介导等温扩增引物的初步筛选可参照该操作手册，具体需要通过实验来验证比较合适的引物。LAMP 引物由 4 个或 6 个(含 Loop)引物组成，建议加入 Loop 环引物设计，即设计 6 条引物进行实验。
2. 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 | (15.6-x) μL | - |
| 10 × Bst 8.0 Reaction Buffer | 2.5 μL | 1 × |
| MgSO₄ (100 mM) | 1.5 μL | 6 mM (8 mM total) |
| dNTP (25 mM each) | 1.4 μL | 1.4 mM each |
| FIP/BIP Primers (25×, 40 μM) | 1 μL | 1.6 μM |
| F3/B3 Primers (25×, 5 μM) | 1 μL | 0.2 μM |
| Loop F/B Primers (25×, 10 μM) | 1 μL | 0.4 μM |
| DNA or RNA Sample | x μL | > 10 copies or more |
| Bst 8.0 DNA Polymerase (40 U/μL) | 1 μL | 1600 U/mL |
| Total volume | 25 μL | - |
注 1：在配制反应体系完成后，可每 25 μL 体系的反应管盖加适量高浓度的 SYBR Green I 1 μL，待等温扩增反应结束后，8000×g 离心 1 分钟，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(4-10 mM)，酶量(0.04-0.32 U/μL)或改变反应温度(50-72 °C)。
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：为了防止在配制试剂时发生污染，请务必在超净工作台内进行操作。
注 7：试剂及模板 DNA 的配制操作尽量需要和 PCR 产物的电泳等分析在不同的区域进行，以免发生污染。
3. 反应程序：65 °C 60 分钟。
4. 灭活：80 °C 5 分钟。
5. 如实验需要，可以用 2.0%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。
保存条件：
-20 °C保存，2 年有效；≤0 °C运输
注意事项：
（1）Bst 8.0 DNA Polymerase 不具有 5'→3'的核酸外切酶活性。建议等温扩增反应温度不能高于 72 °C，否则会导致酶较快速度失活。
（2）Bst 8.0 DNA Polymerase 不能用于热循环测序或 PCR。
（3）每次等温扩增实验建议设置仅无模板 DNA 的阴性对照，以排除背景性扩增。
（4）酶使用时宜存放在冰盒内或冰浴上，使用完毕后宜立即放置于-20 °C 保存。
（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。
Bst 8.0 DNA Polymerase produced by Aladdin is a homolog of Bst DNA Polymerase, large fragment, namely a homolog of the large fragment of DNA polymerase from Bacillus stearothermophilus (Bst). Compared with the large fragment of Bst DNA Polymerase, it exhibits stronger 5'→3' DNA polymerase activity, stronger strand displacement ability, dUTP tolerance, salt tolerance, and tolerance to nonionic detergents. Bst 8.0 DNA Polymerase has neither 5'→3' nor 3'→5' exonuclease activity and can be used for loop-mediated isothermal amplification (LAMP), crossing priming amplification (CPA), rolling-circle amplification (RCA), and isothermal amplification reactions based on rolling-circle amplification. The temperature for isothermal amplification mediated by Bst 8.0 DNA Polymerase is generally between 50-72 °C, usually 65 °C. The optimal temperature depends on the primers used and the amplification products and needs to be optimized experimentally.
| Source | Recombinant expression in E. coli |
| Appearance | Sterile liquid |
| Storage Buffer | 10 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 @ 25 °C). |
| Enzyme Concentration | 40 U/μL |
| Purity | No DNA endonuclease or exonuclease activity. |
| Activity Definition | One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 65 °C. |
Components and Description
| Component | B744265-8KU | B744265-40KU |
|---|---|---|
| Bst 8.0 DNA Polymerase (40 U/μL) | 200 μL | 1 mL |
| 10 × Bst 8.0 Reaction Buffer | 600 μL | 3 mL |
| 100 mM MgSO₄ | 400 μL | 2 mL |
Applications
Loop-mediated isothermal amplification (LAMP), reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reaction temperature up to 72 °C), crossing priming amplification (CPA), rolling-circle amplification (RCA), helicase-dependent amplification (HDA), and other DNA isothermal amplification methods, multiple displacement amplification (MDA), strand displacement DNA synthesis, whole genome amplification (WGA), sequencing of DNA with high GC content, rapid sequencing of nanogram-level DNA templates, library preparation and sequencing, etc.
Product Advantages
This product not only exhibits considerable DNA polymerase activity, strand displacement ability, dUTP tolerance, and high-temperature resistance (tolerating temperatures up to 72 °C), but also possesses better reverse transcriptase activity and stronger dUTP tolerance, making it suitable for reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Instructions for Use
1. Primer Design.
The preliminary screening of primers for loop-mediated isothermal amplification can refer to this instruction manual. Suitable primers need to be verified and compared through experiments. LAMP primers consist of 4 or 6 primers (including Loop primers). It is recommended to include Loop primers in the design, that is, to design 6 primers for the experiment.
2. Taking LAMP isothermal amplification as an example, set up the reaction system according to the following table.
| Reagent | Volume | Final concentration |
|---|---|---|
| Nuclease-free Water | (15.6-x) μL | - |
| 10 × Bst 8.0 Reaction Buffer | 2.5 μL | 1 × |
| MgSO₄ (100 mM) | 1.5 μL | 6 mM (8 mM total) |
| dNTP (25 mM each) | 1.4 μL | 1.4 mM each |
| FIP/BIP Primers (25×, 40 μM) | 1 μL | 1.6 μM |
| F3/B3 Primers (25×, 5 μM) | 1 μL | 0.2 μM |
| Loop F/B Primers (25×, 10 μM) | 1 μL | 0.4 μM |
| DNA or RNA Sample | x μL | > 10 copies or more |
| Bst 8.0 DNA Polymerase (40 U/μL) | 1 μL | 1600 U/mL |
| Total volume | 25 μL | - |
Note 1: After preparing the reaction system, an appropriate amount of high-concentration SYBR Green I (1 μL) can be added to the cap of each reaction tube containing a 25 μL reaction system. After the isothermal amplification reaction is completed, centrifuge at 8000×g for 1 minute. A fluorescent green reaction system indicates a positive result, while a colorless or brown reaction system indicates a negative result. Alternatively, no indicator needs to be added. After the reaction program is completed, an obviously turbid reaction solution indicates a positive result, while a transparent reaction solution indicates a negative result.
Note 2: If reaction optimization is required, the Mg²⁺ concentration (4-10 mM), enzyme amount (0.04-0.32 U/μL), or reaction temperature (50-72 °C) can be adjusted.
Note 3: If analysis is performed by agarose gel electrophoresis or other methods requiring the opening of LAMP reaction containers, please establish a separate analysis area and equipment to avoid contamination.
Note 4: Since the reaction proceeds relatively rapidly, it is recommended to add template DNA last to ensure experimental reproducibility.
Note 5: It is strongly recommended to include a negative control without template to ensure the specificity of amplification.
Note 6: To prevent contamination during reagent preparation, please ensure that operations are performed in a clean bench.
Note 7: Reagent and template DNA preparation should be performed in areas separate from those used for PCR product electrophoresis and other analyses to avoid contamination.
3. Reaction Program: 65 °C for 60 minutes.
4. Inactivation: 80 °C for 5 minutes.
5. If required, electrophoresis analysis can be performed using a 2.0% agarose gel. The appearance of ladder-like bands of amplified products on the electrophoresis gel indicates a positive result, while the absence of ladder-like bands indicates a negative result.
Storage Conditions:
Store at -20 °C, valid for 2 years; transport at ≤0 °C.
Precautions:
(1) Bst 8.0 DNA Polymerase does not possess 5'→3' exonuclease activity. It is recommended that the isothermal amplification reaction temperature should not exceed 72 °C; otherwise, the enzyme will be rapidly inactivated.
(2) Bst 8.0 DNA Polymerase cannot be used for thermal cycling sequencing or PCR.
(3) It is recommended to include a negative control containing no template DNA in each isothermal amplification experiment to exclude background amplification.
(4) The enzyme should be kept in an ice box or on ice during use and immediately stored at -20 °C after use.
(5) This product is intended for scientific research by professionals only. It must not be used for clinical diagnosis or treatment, food or pharmaceutical applications, or stored in ordinary residential premises.
(6) For your safety and health, please wear a laboratory coat and disposable gloves during operation.
规格
别名
Bst 8.0 DNA 聚合酶
规格或纯度
BioReagent, 分子生物学级, 无菌, 40 U/μL
英文名称
Bst 8.0 DNA Polymerase
单位定义
One unit is defined as the amount of enzyme that will incorporate 10nmol of dNTP into acid insoluble material in 30 minutes at 65ºC。
储存与运输
储存条件
-20°C储存
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (24 months).
组分与储存
组件产品号 | 组件名称 | 物理外观 | 8 KU | 40 KU | 储存温度 |
|---|---|---|---|---|---|
B744265-A | Bst 8.0 DNA Polymerase (40 U/μL) | 液体 | 200 μL | 1 mL | -20 °C |
B744265-B | 10 × Bst 8.0 Reaction Buffer | 液体 | 600 μL | 3 mL | -20 °C |
B744265-C | 100 mM MgSO₄ | 液体 | 400 μL | 2 mL | -20 °C |
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