Bst 6.0 DNA Polymerase

Bst 6.0 DNA 聚合酶

Bst 6.0 DNA Polymerase

B744264-8KU

8KU

EnzymoPure™

无DNA内切酶和外切酶活性。

-20°C储存

超低温冰袋运输

-20ºC保存，两年有效。

阿拉丁研发生产的Bst 6.0 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、更强的链置换能力、dUTP耐受性、耐盐性以及非离子去垢剂耐受性。Bst 6.0 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 6.0 DNA Polymerase介导的等温扩增温度一般在50-68ºC之间，通常为65ºC，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。活性定义：One unit is defined as the amount of enzyme that will incorporate 10nmol of dNTP into acid insoluble material in 30 minutes at 65°C。阿拉丁生产的Bst 6.0 DNA Polymerase酶活性效果参考图1。图1. 阿拉丁Bst 6.0 DNA Polymerase 进行环介导等温扩增反应的效果图。在25μl体系，以相同量(0.1ng)的pCMV-Cre-EGFP 为模板，使用不同量的阿拉丁的本产品或N公司(Competitor)的Bst 2.0 DNA polymerase，引物：1.6µM FIP/BIP，0.2µM F3/B3，0.4µM Loop F/B，1.4mM dNTP each，在1X Bst 6.0 Reaction Buffer (2mM MgSO4)中补加MgSO4至终浓度为8mM，65ºC孵育1小时，80ºC加热20分钟灭活，然后进行2.0%的琼脂糖凝胶电泳检测。如图所示，本产品与Competitor N公司的产品相比，具有相当的酶活性效果。-, 仅未添加酶的阴性对照；+, 仅未添加模板的阴性对照; M, DNA marker (DNA Ladder (0.2-12 kb, 12 bands) )。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。阿拉丁生产的Bst 6.0 DNA Polymerase进行环介导等温扩增反应可耐受高浓度的dUTP参考图2。图2. 阿拉丁Bst 6.0 DNA Polymerase 进行环介导等温扩增反应的高dUTP耐受性的效果图。在25μl体系，以相同量(0.1ng)的pCMV-Cre-EGFP 为模板，使用不同量的本产品或N公司(Competitor)的Bst 2.0 DNA Polymerase，引物：1.6µM FIP/BIP，0.2µM F3/B3，0.4µM Loop F/B，1.4mM dNTP each，1.4mM dUTP，0.04U UDGase，在1X Bst 6.0 Reaction Buffer (2mM MgSO4)中补加MgSO4至终浓度为8mM，65ºC孵育1小时，80ºC加热20分钟灭活，然后进行2.0%的琼脂糖凝胶电泳检测。如图所示，本产品与Competitor N公司的产品相比，具有类似的高dUTP耐受性。-, 仅未添加酶的阴性对照；+, 仅未添加模板的阴性对照; M, DNA marker (DNA Ladder (0.2-12 kb, 12 bands) )。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。阿拉丁生产的Bst 6.0 DNA Polymerase进行环介导等温扩增反应可耐受高浓度的非离子去垢剂参考图3。图3. 阿拉丁Bst 6.0 DNA Polymerase 进行环介导等温扩增反应的高非离子去垢剂耐受性的效果图。在25μl体系，以相同量(0.1ng)的pCMV-Cre-EGFP 为模板，使用相同量的本产品或N公司(Competitor)的Bst 2.0 DNA Polymerase，引物：1.6µM FIP/BIP，0.2µM F3/B3，0.4µM Loop F/B，1.4mM dNTP each，5%非离子去垢剂（A, Tween-20; B, NP-40; C, TritonX-100），在1X Bst 6.0 Reaction Buffer (2mM MgSO4)中补加MgSO4至终浓度为8mM，65ºC孵育1小时，80ºC加热20分钟灭活，然后进行2.0%的琼脂糖凝胶电泳检测。如图所示，本产品与Competitor N公司的产品相比，具有类似对非离子洗涤剂的高耐受性。-, 仅未添加酶的阴性对照；+, 仅未添加模板的阴性对照; M, DNA marker (DNA Ladder (0.2-12 kb, 12 bands) )。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。阿拉丁生产的Bst 6.0 DNA Polymerase进行环介导等温扩增反应可耐受高浓度的钾离子(K+)参考图4。图4. 阿拉丁Bst 6.0 DNA Polymerase 进行环介导等温扩增反应的耐盐性(K+)效果图。在25μl体系，以相同量(0.1ng)的pCMV-Cre-EGFP 为模板，使用不同量的本产品或N公司(Competitor)的Bst 2.0 DNA Polymerase，引物：1.6µM FIP/BIP，0.2µM F3/B3，0.4µM Loop F/B，1.4mM dNTP each，在1X Bst 6.0 Reaction Buffer (2mM MgSO4)中补加MgSO4至终浓度为8mM，同时补加KCl至终浓度为10mM、50mM或100mM，65ºC孵育1小时，80ºC加热20分钟灭活，然后进行2.0%的琼脂糖凝胶电泳检测。如图所示，本产品与Competitor N公司的产品相比，具有类似的耐盐性(K+)。-, 仅未添加酶的阴性对照；+, 仅未添加模板的阴性对照; M, DNA marker (DNA Ladder (0.2-12 kb, 12 bands) )。实际操作时不同实验条件获得的实验结果会略有差异，图中所示结果仅供参考。 用途 ： 环介导等温扩增LAMP、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。 来源 ： Bst 6.0 DNA Polymerase通过大肠杆菌重组表达和纯化而获得。 酶储存溶液 ： 10mM Tris-HCl, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 @ 25℃）。10X Bst 6.0 Reaction Buffer: 200mM Tris-HCl, 500mM KCl, 100mM (NH4)2S04, 20mM MgS04, 1%Tween-20 (pH 8.8 @ 25℃)。 失活或抑制 ： 80ºC加热20分钟可使Bst 6.0 DNA Polymerase失活。 注意事项 ： Bst 6.0 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70ºC，否则会导致酶失活。Bst 6.0 DNA Polymerase不能用于热循环测序或PCR。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。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-20ºC保存；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。为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.引物设计。环介导的等温扩增引物的设计可以参考http://primerexplorer.jp/e/，建议使用V5版本。具体操作手册可以在http://primerexplorer.jp/e/v5_manual/index.html下载。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 - 10X Bst 6.0 Reaction Buffer 2.5μl 1X MgSO4 (100mM) 1.5μl 6mM (8mM total) dNTP (25mM each) 1.4μl 1.4mM each FIP/BIP Primers (25X, 40μM) 1μl 1.6μM F3/B3 Primers (25X, 5μM) 1μl 0.2μM Loop F/B Primers (25X, 10μM) 1μl 0.4μM Template xμl ＞ 10 copies or more Bst 6.0 DNA Polymerase (40U/μl) 1μl 1600U/ml Total volume 25μl - 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，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(4-10mM)，酶量(0.04-0.32U/μl)或改变反应温度(50-68ºC)。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：65ºC 60分钟。4.灭活：80ºC 20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。 Aladdin's Bst 6.0 DNA Polymerase is a homolog of Bst DNA Polymerase, Large fragment , which is derived from Bacillus stearothermophilus (Bst). Compared to the large fragment of Bst DNA Polymerase, Bst 6.0 DNA Polymerase has higher 5'→3' DNA polymerization activity, stronger strand displacement ability, tolerance to dUTP, salt, and non-ionic detergents. This product does not possess 5'→3' and 3'→5' exonuclease activities and can be used in various applications such as loop-mediated isothermal amplification (LAMP), crossing priming amplification (CPA), rolling circle amplification (RCA), and isothermal amplification reactions based on RCA. Its optimal working temperature for isothermal amplification is generally between 50-68℃, typically at 65℃, which is dependent on the primers and template DNA used, and needs to be optimized through experiments.Unit definition Application ： Loop-mediated isothermal amplification (LAMP), cross-primer amplification (CPA), rolling circle amplification (RCA), helicase isothermal amplification (HDA), and other isothermal amplification of DNA, multiple-displacement amplification (MDA), strand-displaced DNA synthesis, whole-genome amplification (WGA), high GC-content DNA sequencing, rapid sequencing of nanogram-level DNA templates, library preparation for DNA sequencing, etc.Enzyme storage buffer: 10mM Tris-HCl, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 at 25℃）10X Bst 6.0 Reaction Buffer: 200mM Tris-HCl, 500mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1%Tween-20 (pH 8.8 at 25℃).Denaturation or inactivation: Bst 6.0 DNA Polymerase can be inactivated by heating at 80°C for 20 minutes. Source ： Recombinantly expressed and purified from E. coli.Purity: Free of DNA endonuclease and exonuclease.Applications: Loop-mediated isothermal amplification (LAMP), cross-primer amplification (CPA), rolling circle amplification (RCA), helicase isothermal amplification (HDA), and other isothermal amplification of DNA, multiple-displacement amplification (MDA), strand-displaced DNA synthesis, whole-genome amplification (WGA), high GC-content DNA sequencing, rapid sequencing of nanogram-level DNA templates, library preparation for DNA sequencing, etc. Enzyme storage buffer ： 10mM Tris-HCl, 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% Glycerol (pH 7.5 at 25℃）10X Bst 6.0 Reaction Buffer: 200mM Tris-HCl, 500mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1%Tween-20 (pH 8.8 at 25℃).Denaturation or inactivation: Bst 6.0 DNA Polymerase can be inactivated by heating at 80°C for 20 minutes. Precautions ： Bst 6.0 DNA Polymerase does not possess 5'-＞3' exonuclease activity.The temperature for isothermal amplification should not exceed 70°C. Otherwise, the enzyme will be inactivated.Bst 6.0 DNA Polymerase cannot be used for thermal cycle sequencing or PCR.Always set up a negative control without template DNA for each isothermal amplification experiment to exclude background amplification.This product should be kept on ice during use, and stored at -20°C immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation Instructions for Use ： 1. Primer designFor the design of loop-mediated isothermal amplification primers, please refer to http://primerexplorer.jp/e/ and version V5 is recommended. The manual can be downloaded at http://primerexplorer.jp/e/v5_manual/index.html. Refer to this manual for the preliminary screening of loop-mediated isothermal amplification primers (LAMP), and more suitable primers need to be verified by experiments. LAMP primers consist of 4 or 6 (including Loop) primers. It is recommended to design 6 primers for the experiment.2. Set up LAMP reactions on ice as follows:ReagentVolumeFinal concentrationNuclease-free Water (, ST876)(15.6-x)μl-10X Bst 6.0 Reaction Buffer2.5μl1XMgSO4 (100mM) 1.5μl6mM (8mM total)dNTP (25mM each) (, D7373)1.4μl1.4mM eachFIP/BIP Primers (25X, 40μM)1μl1.6μMF3/B3 Primers (25X, 5μM)1μl0.2μMLoop F/B Primers (25X, 10μM)1μl0.4μMTemplatexμl＞ 10 copies or moreBst 6.0 DNA Polymerase (40U/μl)1μl1600U/mlTotal volume25μl-Note 1: After preparing the reaction, add 1μl of high-concentration SYBR Green I to each tube. After isothermal amplification, centrifuge at 8,000×g for 1 min, positive reactions turn fluorescent green, while negative reactions remain colorless or brown. Even without any indicators, positive reactions can be identified by the turbidity of reactions. Negative reactions are clear and transparent.Note 2: To optimize the reaction, adjust the Mg2+ concentration (4-10mM), the amount of enzyme (0.04-0.32U/μl) or change the reaction temperature (50-68℃).Note 3: If analysis is performed by agarose gel electrophoresis or other methods that require opening of the LAMP reaction vessel, set up auxiliary analysis areas and equipment to avoid contamination.Note 4: In order to ensure the reproducibility of the experiment, it is recommended to add template DNA at the last.Note 5: It is strongly recommended to set a negative control without template to confirm the specificity of amplifications.Note 6: To prevent contamination, the reaction mixture should be prepared on a clean bench.Note 7: The preparation of reagents and template DNA should be performed in different areas from the electrophoresis area to avoid contamination.3. Incubate at 65℃ for 60 minutes.4. Incubate at 80℃ for 20min to terminate the reaction.5. If necessary, examine the reaction products by 2.0% agarose gel electrophoresis. The amplification products should show a ladder-like band pattern.