EnzymoPure™ pG-Tn5 Transposase

EnzymoPure™ pG-Tn5 转座酶

EnzymoPure™ pG-Tn5 Transposase

P745697-4000pmol

4000pmol

EnzymoPure™

-20°C储存

超低温冰袋运输

-20℃保存，一年有效。

阿拉丁生产的EnzymoPure™ pG-Tn5 Transposase，中文名为EnzymoPure™ pG-Tn5转座酶，是一种来源于E.coli，经过改造的具有极高活性Tn5转座酶突变体和Protein G的融合蛋白。pG-Tn5转座酶在和适当测序接头(Adapters)组装后成为pG-Tn5 Transposome (pG-Tn5转座体)，可用于CUT&Tag实验。CUT&Tag (Cleavage Under Targets and Tagmentation)，即靶向剪切及标记，是一种新兴的蛋白质与DNA相互作用的研究方法，具有特异性强、背景噪音较低、灵敏度高、重复性较好、成本低等优点，被用来替代传统的ChIP-Seq (Chromatin Immunoprecipitation-sequencing)方法。CUT&Tag技术的核心是利用pA/G-Tn5 Transposome上的Protein A或Protein G具有与靶蛋白上结合的抗体特异性结合的能力，使Tn5转座体能通过Protein A/G及抗体与靶蛋白形成复合物，随后加入Mg2+激活Tn5转座体的酶活性对靶蛋白结合的DNA进行切割实现靶向的DNA片段化(DNA Fragmentation)，同时将其携带的测序接头连接到片段两端从而实现标记，后续通过PCR反应进行缺口填补，并借助双端标记PCR引物(Dual-indexed PCR primers)扩增后，制备成适用于二代测序(Next generation sequencing, NGS)平台的DNA文库 1]。EnzymoPure™ pG-Tn5 Transposase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。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。 注意事项 ： 本品含50%甘油，-20℃保存不会冻结。须避免-80℃保存，否则会冻结， 反复冻融可能会降低本产品的酶活性。本品较为粘稠，吸取时注意取样量准确，加样后请注意充分吹打混匀，避免产生气泡。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 ： 构建CUT&Tag实验所需的 pG-Tn5转座体。1.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。2.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 使用PCR仪按下表设置退火反应程序，反应结束即可获得Adapter 1和Adapter 2。 Step Temperature 1 95℃, 2min 2 95℃, 8s, -0.1℃ per cycle 3 GOTO step 2700 cycles 4 4℃ forever 3. pG-Tn5转座体的制备。按照下表，将 pG-Tn5 Transposase、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。 Component Volume pG-Tn5 Transposase (40μM) 10μl Adaptor 1 (200μM) 1μl Adaptor 2 (200μM) 1μl 注：可根据实验需要适当提高 pG-Tn5 Transposase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CUT&Tag实验，也可以-20℃保存备用。参考文献：1. Hatice S. Nat Commun. 2019.10: 1930. EnzymoPure™ pG-Tn5 Transposase is an E.coli-derived fusion protein of mutated Tn5 transposase and protein G, with high Tn5 transposase activity. The pG-Tn5 Transposome comprising the pG-Tn5 transposase and appropriate sequencing adapters can be used for CUT&Tag assays.CUT&Tag (Cleavage Under Targets and Tagmentation) is an emerging approach for protein-DNA interaction studies with the advantages of high specificity, low background noise, high sensitivity, good reproducibility and low cost. It has been used as a substitute of the traditional ChIP-Seq (Chromatin Immunoprecipitation-sequencing) in protein-DNA interaction studies. The CUT&Tag technique utilizes the ability of Protein A or Protein G on the pA/G-Tn5 Transposome to specifically bind antibodies of the target protein, enabling the Tn5 transposome to bind the target protein. Subsequently, Mg2+ is added to activate Tn5 Transposase which cleaves the target protein bound DNA into fragments and simultaneously attaches the sequencing adapters to both ends of the DNA fragments. After PCR amplification with Dual-indexed PCR primers, the DNA library is ready for Next Generation Sequencing (NGS) [1].EnzymoPure™ pG-Tn5 Transposase is a recombinant protein expressed in E.coli and purified by Aladdin's proprietary PerfectProtein™ Technology Platform. Compared with wild-type Tn5 transposase, this product has at least 1000-fold higher insertion efficiency, with good transposition randomness, high stability and easy identification of the insertion site.Enzyme storage solution Precautions ： This product contains 50% glycerol and will not freeze when stored at -20ºC. Avoid storing at -80ºC as it will freeze and repeated freeze-thaw may reduce the enzymatic activity of the product. This product is viscous. Be careful to take the correct amount when aspirating and avoid the formation of air bubbles when mixing it with samples.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 ： Construction of the pG-Tn5 transposon required for CUT&Tag experiments.1.Sequence of MEs and Adapters.ME: 5'-[phos]CTGTCTCTTATACACATCT-3'Primer-1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'Primer-2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'Note: This adapter sequence is referenced to the Nextera® DNA Sample Preparation Kit (Illumina, FC-121-1030) and can also be adjusted to meet the requirements of different high-throughput sequencing platform.2.Preparation of Adapter 1 and Adapter 2.Set up the following annealing reactions for Adapter 1 and Adapter 2, respectively. The final concentration of Adapter 1 or Adapter 2 in the reaction is 200μM. Component Volume Annealing Buffer for DNA Oligos (5X) 10μl ME (500μM) 20μl Primer-1 or Primer-2 (500μM) 20μl Total Reaction Volume 50μl Transfer the reactions onto a thermal cycler and run the thermocycling conditions as below: Step Temperature 1 95ºC, 2min 2 95ºC, 8s, -0.1ºC per cycle 3 GOTO step 2700 cycles 4 4ºC forever 3.Preparation of the pG-Tn5 transposome.Mix pG-Tn5 Transposase, Adapter 1 and Adapter 2 in a molar ratio of 1:0.5:0.5 according to the table below, mix well by pipetting, and incubate for 1 hour at room temperature. Component Volume pG-Tn5 Transposase (40μM) 10μl Adaptor 1 (200μM) 1μl Adaptor 2 (200μM) 1μl Note: The amount of pG-Tn5 Transposase in the mix can be increased if required, but the concentrations of Adapter 1 and Adapter 2 must be the same. The prepared pG-Tn5 transposome can be used directly in CUT&Tag experiments or stored at -20ºC for future use.References:1.Hatice S. Nat Commun. 2019.10: 1930.