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| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| T745702-800pmol |
800pmol |
期货  |
| |
| T745702-4000pmol |
4000pmol |
期货 |
|
| 稳定性与储存 | -20℃保存，一年有效。 |
|---|---|
| 英文名称 | EnzymoPure™ Tn5 Transposase |
| 储存温度 | -20°C储存 |
| 运输条件 | 超低温冰袋运输 |
| 产品介绍 |
产品介绍： 阿拉丁生产的EnzymoPure™ Tn5 Transposase，中文名为EnzymoPure™ Tn5转座酶，是一种来源于E.coli、经过改造具有极高活性的Tn5转座酶突变体，可以高效地将Tn5转座子(Transposon)随机插入到目标序列，对于真核和原核生物的DNA都有极高的转座插入效率。本产品可以特异性识别两端含有嵌合末端序列(Mosaic End sequence, ME)的DNA片段(包括含有ME序列的引物)，最终形成Tn5转座体(Tn5 Transposome)，该转座体可以随机结合靶DNA并切割插入其携带的DNA片段。Tn5转座酶被广泛用于体外转基因(外源基因整合到宿主细胞)和二代测序(Next Generation Sequencing, NGS)建库等领域。转座子(Transposon)，也称为转座元件(Transposable elements, TEs)、跳跃基因(Jumping genes)，是一种可以在基因组中“跳跃”到不同位置的遗传元件(Genetic elements)。尽管转座子经常被称为跳跃基因，转座子还是会相对比较稳定地存在于相应的整合位点，并且大部分转座子最终会变得没有跳跃活性。转座子最早由Barbara McClintock在玉米中发现，并在1983年获得了诺贝尔生理医学奖。后续研究发现转座子系统广泛存在于生物界，并逐渐研究开发成为基因分析的有效工具。EnzymoPure™ Tn5转座酶是由阿拉丁自主研发的PerfectProtein™技术平台表达、纯化获得的重组蛋白，与野生型Tn5转座酶相比，其插入效率至少提升1000倍，具有转座随机性好、稳定性高、插入位点易测序等特点。图1. 阿拉丁的EnzymoPure™ Tn5 Transposase的工作原理图。EnzymoPure™ Tn5 Transposase与Tn5 Transposase的工作原理相同。Tn5转座酶的工作原理如图1所示。当转座事件发生时，两个Tn5转座酶分子结合到供体DNA (Donor DNA)的转座子的ME序列，形成两个Tn5转座酶分子与供体DNA的复合物①，随后两个Tn5转座酶分子通过C末端相互作用进行联会并环化(Circularization)转座子，形成一个Tn5转座酶二聚体复合物 (Transposase dimer complex)②，在Mg2+存在的条件下，Tn5转座酶切割供体DNA，并携带供体DNA片段离开供体链形成转座体(Transposome)③，当Tn5转座体识别靶点DNA (Target DNA)，也称受体DNA ，并结合到随机靶序列(Target site)上形成转座体与靶序列的复合物④，在Mg2+存在条件下，会对靶序列进行切割，将其携带的供体DNA片段插入靶序列中，形成转座后的DNA序列(Transposed DNA)⑤，切割形成的9bp粘性末端可以通过DNA聚合酶、连接酶作用进行填补，最终两端形成9bp正向重复序列。基因从供体DNA通过Tn5转座酶“剪切”之后“粘贴”到另一受体DNA，实现了基因片段在基因组中的“跳跃”。产品用途： 本产品可用于二代测序(NGS)文库构建时的片段化和加接头；将测序引物引入克隆DNA或质粒；细菌基因敲除库的建立；新型细菌菌株工程改造；目的基因的插入失活；将T7转录启动子、抗性标记等插入靶DNA等。酶储存溶液： 50mM HEPES (pH7.2), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) Glycerol。Reaction Buffer (5X)：50mM HEPES (pH7.2), 500mM NaCl, 50mM MgCl2。Stop Buffer (5X)：50mM EDTA (pH8.0)。用于体外转座子插入反应时，按每次使用1μl Tn5 Transposase (40μM)，本产品小包装D7102S可以进行20次反应，中包装D7102M可以进行100次反应。使用说明： 1.体外转座子插入反应和转化。a.Tn5转座子的设计。Tn5转座子是两侧带有19bp ME序列的DNA片段，可以使用含有ME序列的上下游引物进行PCR合成。为了获得最佳的转座效率，在两端的PCR引物中需要添加一个5'磷酸基团。典型的Tn5转座子参考图2。ME序列: 5'-[phos]CTGTCTCTTATACACATCT-3'图2. Tn5转座子示意图。Tn5转座子其两端为19bp ME序列，ME序列可被 Tn5转座酶特异性识别。b.按照下表制备转座子插入混合物。配制过程中需要注意目标DNA纯度，确保无外源核酸污染。 Component Volume Reaction Buffer (5X) 2μl Target DNA* 0.2μg molar equivalent Tn5 Transposon x μl Tn5 Transposase (40μM) 1μl ddH2O To 10μl *注：为了避免多余片段的插入，需要计算反应中使用的目标DNA摩尔数，并加入等摩尔量的Tn5转座子片段以减少产生过多插入的可能性。μmol target DNA=μg target DNA/[(base pairs in target DNA)×660]，例如：0.2μg 5000bp的目标DNA = 0.2μg/ [5000bp×660]=0.06×10-6μmol = 0.06pmol。c.混匀后37℃孵育2小时。d.加入2μl Stop Buffer (5X)，混匀后55℃孵育10分钟终止反应。e.取1μl混合物电击转化到感受态细胞中，根据抗性标记筛选阳性菌株。未使用的混合物可以-20℃保存。推荐的电击转化条件：50μl感受态细胞，1μl混合物，2毫米电击杯，2500V，电击时间5毫秒。转化条件可根据该条件的转化效果进行优化。转座的克隆数主要取决于所转化的宿主细胞、内源性的限制修饰系统及感受态细胞的转化效率。2. Tn5转座体复合物( Tn5 Transposome complex)的构建和转化后的体内插入。a.按照下表依次加入相应试剂，制备Tn5转座体复合物混合物。 Component Volume Tn5 Transposon DNA (100μg/ml in TE Buffer) 2μl Tn5 Transposase (40μM) 4μl Glycerol (100%) 2μl Total Reaction Volume 8μl 注1：本反应无须Mg2+催化，请勿使用D7102-2 Reaction Buffer (5X)。注2：参考1a中Tn5的转座子设计，Tn5 Transposon DNA为含选择标记(如抗性标记)、成对识别序列(如ME序列)和目标基因的双链DNA，可通过PCR等方法获得。注3：本反应体系可根据实际需要进行放大或缩小。b.混匀后室温孵育0.5-1小时。c.取1μl的转座体复合物电击转化到感受态细胞中，在体内进行插入，并根据抗性标记筛选阳性菌株。推荐的电击转化条件：50μl感受态细胞，1μl转座体复合物 ，2毫米电击杯，2500V，电击时间5毫秒。转化条件可根据该条件的转化效果进行优化。转座的克隆数主要取决于所转化的宿主细胞、内源性的限制修饰系统及感受态细胞的转化效率。d.构建好的转座体复合物-20℃可以保存1年。3.构建二代测序所需的 Tn5转座体( Tn5 Transposome)。a.ME和接头(Adapters)的序列。ME: 5'-[phos]CTGTCTCTTATACACATCT-3'Primer 1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'Primer 2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'注：本接头序列参考Nextera® DNA Sample Preparation Kit (Illumina, FC-121-1030)，也可按照不同高通量测序的要求进行设计。b.制备Adapter 1和Adapter 2。按照下表分别配制Adapter 1和Adapter 2退火反应体系。退火反应后Adapter 1或Adapter 2的终浓度为200μM。 Component Volume Annealing Buffer for DNA Oligos (5X) 10μl ME (500μM) 20μl Primer-1 or 2 (500μM) 20μl Total Reaction Volume 50μl 在PCR仪设置退火反应程序： Step Temperature 1 95℃, 2min 2 95℃, 8s, -0.1℃ per cycle 3 GOTO step 2, 700 cycles 4 4℃ forever c. Tn5转座体的制备。按照下表，将 Tn5转座酶、Adapter 1和Adapter 2按摩尔比1:0.5:0.5混合，吹打混匀，室温孵育1小时。注：可根据实验需要适当提高Tn5转座酶的混合比例，但Adapter 1和Adapter 2的浓度需要保持一致。制备好Tn5转座体可直接用于DNA片段化实验，也可以-20℃保存。 Component Volume Tn5 Transposase 10μl Adapter 1 (200μM) 1μl Adapter 2 (200μM) 1μl d.片段化(Tagmentation)效果测试。按照下表配制反应体系，轻轻吹打混匀后，55℃孵育5-10分钟。随后加入5μl Stop Buffer (5X)，混匀后55℃孵育5分钟终止反应，片段化的产物可用于检测或纯化后建库, 效果参考图3。根据打断片段的大小调整复合体用量，如果片段过长，增加转座体的使用量；如果片段过短，则减少转座体的使用量。 Component Volume DNA 50-100ng Tn5转座体 0.5-2μl Reaction Buffer (5X) 4μl ddH2O To 20μl 图3. 使用阿拉丁的 Tn5 Transposase制备的Tn5转座体用于DNA随机片段化的效果测试图。在20μl反应体系中，加入100ng Lambda DNA及相应量的Tn5转座体，55℃孵育10分钟，反应完毕入5μl Stop Buffer (5X)，混匀后55℃孵育5分钟终止反应，加入5μl DNA上样缓冲液(6X) ，使用 琼脂糖预制胶进行电泳检测。实际效果会因样品种类、检测仪器等的不同而存在差异，图中数据仅供参考。Aladdin's EnzymoPure™ Tn5 Transposase is a hyperactive, mutated form of Tn5 Transposase and a highly efficient enzyme for insertion of a Tn5 transposon into both eukaryotic and prokaryotic DNA. This product specifically recognizes DNA fragments containing Mosaic End (ME) sequences at each of its ends (including primers containing ME sequences), resulting in the formation of a Tn5 transposome which randomly attacks and cleaves the target DNA to insert the DNA fragment it carries.EnzymoPure™ Tn5 Transposase is recombinantly expressed in E.coli and purified on Aladdin's PerfectProtein™ technology platform. It possesses good randomness, high stability, and a transposition frequency that is 1000-fold greater than wild type Tn5 transposase.Figure 1. Schematic diagram of transposition reaction catalyzed by Aladdin's EnzymoPure™ Tn5 Transposase .EnzymoPure™ Tn5 Transposase catalyzes a multi-step “cut and paste” transposition reaction (Figure 1). Initially, the enzyme binds the ME sequence of the transposon to form a complex①. Then two Tn5 transposases at two ends of one transposon associate and circularize the transposon through C-terminal interactions, forming a Transposase dimer complex②. In the presence of Mg2+, the Tn5 transposase cleaves the donor DNA and carries the donor DNA fragment to form the Transposome③. The Tn5 transposome recognizes and binds the target DNA to form a complex of transposome and target sequence④. In the presence of Mg2+, the Tn5 transposase cuts the target sequence and insert the donor DNA fragment it carries into the target sequence, forming the transposed DNA⑤. Finally, the 9bp sticky ends created by transposition can be blunted by DNA polymerase and ligase, forming a 9bp sequence duplication immediately flanking the transposon insertion site.s Application： Fragmentation and tagmentation during next-generation sequencing (NGS) library preparation; introduction of sequencing primers into cloned DNA or plasmids; construction of bacterial gene knockout libraries; engineering of novel bacterial strains; insertional inactivation of target genes; insertion of T7 promoter and resistance marker into target DNA.Storage Buffer: 50mM HEPES (pH7.2), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) Glycerol.Reaction Buffer (5X): 50mM HEPES (pH7.2), 500mM NaCl, 50mM MgCl2.Stop Buffer (5X): 50mM EDTA (pH8.0).For in vitro transposition, use 1μl of Tn5 Transposase (40μM) per reaction. D7102S and D7102M are sufficient for 20 and 100 reactions, respectively.Precautions： This product contains 50% glycerol and does not freeze at -20ºC. Store this product at -20ºC, but not -80ºC to avoid repeated freeze-thaws.This product is viscous, so be careful to take the right amount when aspirating. After adding to samples, mix well thoroughly by pipetting and avoid the formation of air bubbles. Tn5 Transposase uses DNA as substrate, but not RNA.This product is only applicable to bacteria for transposon insertion. Eukaryotic cells are difficult for the insertion of transposon due to the blockage by nuclear membrane. Please optimize the reaction conditions according to the relevant literature.For bacteria related studies, please do not use chemically competent cells. It is recommended to use electrocompetent cells with the electrotransformation efficiency higher than 106cfu/µg.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. In vitro transposon insertiona. Generation of Tn5 transposon. The Tn5 transposon is a DNA fragment consisting of target DNA sequence flanked by the 19bp ME sequence at both ends, and can be synthesized by PCR using primers that have non-homologous tails with 19-base ME sequnces at 5’-ends and homologous sequences to the template at 3’-ends. For optimal transposition efficiency, primers needs to be 5' phosphated. Please refer to Figure 2 for a typical Tn5 transposon.ME sequence: 5'-[phos]CTGTCTCTTATACACATCT-3'Figure 2. Schematic diagram of Tn5 transposon. The ME sequences at both ends is uniquely and specifically recognized by NGSTM T5 transposase (, D7102)b. Prepare the reaction mix as follows:ComponentQuantityReaction Buffer (5X)2μlTarget DNA*0.2μgEquivalent Moles of Tn5 Transposonx μl Tn5 Transposase (40μM)1μlddH2Oo 10μl*Note: Calculate the moles of target DNA in the reaction and add equivalent moles of Tn5 Transposon to avoid multiple insertions. Target DNA (μmol) = Target DNA (μg)/[(base pairs in target DNA)×660]. For example, the moles of 0.2μg 5000bp target DNA is 0.2μg/ [5000bp×660]=0.06×10-6μmol = 0.06pmol.c. Mix well, incubate at 37ºC for 2 hours.d. Add 2μl of Stop Buffer (5X), mix well and incubate at 55ºC for 10 minutes.e. Electroporate competent cells with 1μl of reaction mix and plate on selective media. The remaining of reaction mix can be stored at -20ºC. The recommended electroporation conditions: 50μl competent cells, 1μl reaction mix, 2mm electroporation cuvette, 2500V, and 5ms. The electroporation conditions can be optimized based on the result. The number of transposed clones depends mainly on the endogenous restriction modification system, competent cell type, and the transformation efficiency of competent cells. 2. Production of Tn5 transposome and in vivo insertiona. Prepare the reaction mix by adding reagents in the following order:ComponentQuantityTn5 Transposon DNA (100μg/ml in TE Buffer)2μl Tn5 Transposase (40μM)4μlGlycerol (100%)2μlTotal Reaction Volume8μlNote 1: This reaction does not need the presence of Mg2+. Do not use the Reaction Buffer (5X).Note 2: Design the Tn5 Transposon according to 1a. A custom Tn5 Transposon consisting of selective marker, a pair of recognition sequences (e.g., ME sequences) and a double-stranded target DNA fragment can be generated by PCR. Note 3: This reaction can be scaled up or down as needed.b. Mix well, incubate at room temperature for 0.5-1h.c. Electroporate competent cells with 1μl of reaction mix and plate on selective media. The recommended electroporation conditions: 50μl competent cells, 1μl reaction mix, 2mm electroporation cuvette, 2500V, and 5ms. The electroporation conditions can be optimized based on the result. The number of transposed clones depends mainly on the endogenous restriction modification system, competent cell type, and the transformation efficiency of competent cells.d. The generated transposome can be stored at -20ºC for up to 1 year.3. Preparation of Tn5 Transposome for NGS a. Sequences of ME and AdaptersME: 5'-[phos]CTGTCTCTTATACACATCT-3'Primer 1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'Primer 2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'Note: The above adaptor sequences are for Nextera® DNA Sample Preparation Kit (Illumina, FC-121-1030). The sequences can also be designed to meet other requirements. b. Preparation of Adapter 1 and Adapter 2Set up the annealing reaction for Adapter 1 and Adapter 2. The final concentration of Adapters after annealing should be 200μM.ComponentQuantityAnnealing Buffer for DNA Oligos (5X) 10μlME (500μM)20μlPrimer-1 or 2 (500μM)20μlTotal Reaction Volume50μlTransfer the reaction into a thermal cycler and run the following annealing conditions:StepTemperature195ºC, 2min295ºC, 8s, -0.1ºC per cycle3GOTO step 2, 700 cycles44ºC foreverc. Production of Tn5 TransposomeAccording to the table below, mix Tn5 transposase, Adapter 1, and Adapter 2 at a molar ratio of 1:0.5:0.5. Mix well by pipetting and incubate at room temperature for 1 hour. The Tn5 transposome can be used directly for DNA tagmentation, or can be stored at -20ºC. Note: The ratio of Tn5 transposase can be increased appropriately according to the experimental requirements, but the concentration of Adapter 1 and Adapter 2 should be the same. ComponentQuantity Tn5 Transposase 10μlAdapter 1 (200μM)1μlAdapter 2 (200μM)1μld. Test of Tagmentation Effect Set up the reaction according to the table below, mix gently by pipetting, and incubate at 55ºC for 5-10 minutes. Stop the reaction by adding 5μl of Stop Buffer (5X) and incubating at 55ºC for 5 minutes. The fragmentation products can be examined or purified for library preparation, as shown in Figure 3. The amount of transposome can be adjusted according to the size of fragments. Increase the amount of transposome if the fragment is too long, and decrease the amount of transposome if the fragment is too short. ComponentQuantityDNA50-100ngTn5 Transposome0.5-2μlReaction Buffer (5X)4μlddH2OTo 20μlFigure 3. Lambda DNA fragmentation by the Tn5 transposome produced by Tn5 Transposase . Reactions of 20μl containing 100ng Lamda DNA and different amounts of Tn5 transposome as shown in the figure were incubated at 55ºC for 10 minutes, followed by the addition of Stop Buffer and incubation at 55ºC for 5 minutes to terminate the reaction. The reaction products were mixed with DNA Loading Buffer (6X) (, D0071) and separated on Agarose Precast Gel . This figure is for reference only, which may vary due to different experimental conditions. |
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