T7 DNA Ligase
规格
| Cas Number | |
| 规格或纯度 | 不含除T7 DNA Ligase之外的其它种类的DNA连接酶，不含DNA内切酶和外切酶，不含RNA酶，不含磷酸酯酶。 |
| 包装 | 3000KU 或 600KU 或 150KU |
产品信息
| 品牌 | 阿拉丁 |
| 浓度 | 不含除T7 DNA Ligase之外的其它种类的DNA连接酶，不含DNA内切酶和外切酶，不含RNA酶，不含磷酸酯酶。 |
| 单位定义 | One unit is defined as the amount of enzyme required to give 50% ligation of 100ng HindIII fragments of λ DNA in a total reaction volume of 20μl in 30 minutes at 25℃ in 1X T7 DNA Ligase Reaction Buffer. |
| 稳定性和储存 | -20℃保存，两年有效。 |
| 过滤标签 | DNA连接 |
| 储存温度 | -20°C储存 |
| 运输条件 | 超低温冰袋运输 |
| 描述 |
阿拉丁生产的T7 DNA Ligase，即T7 DNA连接酶，是由阿拉丁自主研发的PerfectProtein™技术平台表达、纯化获得的一种来源于T7噬菌体的、ATP依赖的双链DNA连接酶，并且对于粘性末端的连接效率远远高于平末端。与T3和T4 DNA Ligase不同，T7 DNA Ligase仅可催化双链DNA相邻粘性末端5'磷酸和3'羟基磷酸二酯键的形成，但不能有效催化平末端双链DNA的连接，加入大于或等于20%的PEG6000可以适当提高该酶的平末端DNA连接活性。因此在平末端和粘性末端双链DNA底物同时存在，且仅需连接粘性末端双链DNA的分子生物学实验中，T7 DNA Ligase是理想的选择1,2]。阿拉丁生产的T7 DNA Ligase用于进行双链DNA粘性末端连接的效果参考图1。图1. 阿拉丁生产的T7 DNA Ligase用于进行双链DNA粘性末端连接的效果图。A. 阿拉丁生产的T7 DNA Ligase与N公司(Competitor)催化产生的重组连接产物转化DH5α感受态后涂LB平板的实测效果图。利用阿拉丁生产的EnzymoPure™ DNA Polymerase ，使用一端带有Hind III酶切识别位点，另一端带有Xba Ⅰ酶切识别位点的引物对靶基因进行PCR扩增，随后使用阿拉丁Hind Ⅲ / Xba Ⅰ对1kb的PCR产物进行双酶切，获得两端带有粘性末端的双链线性DNA分子，作为T7 DNA Ligase催化连接反应的底物。在20µl反应体系(66mM Tris-HCl, 10mM MgCl2, 1mM DTT, 1mM ATP, 7.5% Polyethylene glycol (PEG6000), PH7.6 @25℃)中，分别加入50ng经PCR扩增及Hind Ⅲ/Xba Ⅰ双酶切产生的两端带有粘性末端的双链DNA片段，和经Hind Ⅲ/Xba Ⅰ双酶切线性化的pUC18载体混合(PCR片段与pUC18载体的摩尔比为3:1)，加入10µl的2X Reaction Buffer以及1μl的本产品或N公司(Competitor)的T7 DNA Ligase，然后用水补至20µl，25℃孵育30分钟进行连接。反应结束后，取5µl连接产物转化DH5α超级感受态细胞。B. 菌落PCR鉴定T7 DNA Ligase重组连接构建得到的克隆。实验结果表明，本产品与N公司的产品克隆阳性率一致，具有相当的连接粘性末端双链线性DNA的效果。菌落PCR使用的是pUC18的通用测序引物M13 forward sequencing primer(5´-GTAAAACGACGGCCAGT-3´)和M13 reverse sequencing primer (5´-CAGGAAACAGCTATGAC-3´)。本图仅供参考，实际检测效果可能有所不同。 用途： 限制性内切酶切DNA片段的克隆，双链DNA和接头的连接，线性双链DNA的环化，双链DNA的缺刻修复、定点突变和Transcription Activator-Like Effector Nucleases(TALEN)的DNA片段的Golden Gate Assembly、粘端特异性连接等。来源： 纯化自携带编码T7噬菌体DNA ligase的E.coli重组菌株。酶储存溶液： 10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH7.4 @25℃)。2X Reaction Buffer:132mM Tris-HCl, 20mM MgCl2, 2mM DTT, 2mM ATP, 15% Polyethylene glycol (PEG6000) (pH7.6 @25℃)。失活或抑制： 在不含有PEG6000的反应体系中65℃孵育10分钟。注意事项： ATP是T7 DNA Ligase发挥催化活性所必需的辅助因子，而不是像E.coli DNA Ligase以NAD作为辅助因子的。T7 DNA Ligase不能有效催化平末端双链DNA片段的连接，对于平末端双链DNA的连接建议使用T4 DNA Ligase。T7 DNA Ligase的催化底物是双链DNA，不能用于单链DNA或RNA的连接反应。T7 DNA Ligase的反应体系中含有7.5% PEG6000。如果实验体系中不能加入PEG6000，可考虑自行配制不含PEG6000的连接反应缓冲液，或者使用T4 DNA Ligase的连接缓冲体系，但T7 DNA Ligase连接酶在T4 DNA Ligase的连接缓冲体系中的活性会降低约10倍。反应体系所需的超纯水推荐使用 Ultrapure Water (DNase/RNase-Free, Sterile) 。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： 1.参考下表在冰浴中配制反应体系(以20μl体系为例)： Reagent Volume 2X Reaction Buffer 10µl Vector DNA Xμl (0.020pmol) Insert DNA Yμl (0.060pmol) Ultrapure Water (9-X-Y)μl T7 DNA Ligase (3KU/μl) 1µl Total Volume 20µl 注1：建议按照DNA片段与线性化载体摩尔比3:1的比例进行连接反应。注2：T7 DNA Ligase建议最后添加。2.移液器吹打混匀，低速离心使粘附在管壁上的液体沉降至管底。3.反应条件：25℃(或室温)孵育15~30分钟。4.反应完成后需将连接产物立即置于冰上，取5μl左右连接产物转化到50μl感受态细胞中。剩余样品可选择性的保存于-20℃。注1：不能进行热失活，热失活会显著降低连接产物的转化效率。注2：为检测连接效率，也可将反应后的产物进行琼脂糖凝胶或聚丙烯凝胶电泳，拍照观察并分析连接效果。如果需要从琼脂糖凝胶中回收DNA样品，推荐使用D0056 DNA凝胶回收试剂盒； 常见问题：1.T7 DNA Ligase只能催化粘性末端双链DNA分子的连接。那么T7 DNA Ligase可以连接多少长度的粘性末端?T7 DNA Ligase可以有效催化2bp或更长粘性末端的连接，不能连接1bp的粘性末端。通常情况下，T7 DNA Ligase一般不能连接平末端双链DNA；但当反应体系中含有非常高浓度的PEG6000 (20-30% w/v)时，T7 DNA Ligase对平末端双链DNA也具有一定的连接活性。2.T7 DNA Ligase可以和不含PEG6000的缓冲液一起使用吗?可以。如果实验体系中不能加入PEG6000，我们推荐自行配制仅去除了PEG6000的2X Reaction Buffer。3.使用T7 DNA Ligase进行连接反应时，有哪些潜在的因素会导致转化失败?有以下因素会导致连接反应失败：a.反应体系中缺少ATP或Mg2+会导致连接失败。 缓冲液中的ATP，保存时间过长可能会逐渐降解导致此问题的发生。建议使用新鲜提供的缓冲液或适量补充ATP到反应体系中，以保证连接效率。b.反应体系中存在高盐或EDTA会导致连接失败。建议纯化连接底物，去除干扰。c.CIP, BAP或SAP等磷酸酶在去磷酸化过程中没有完全失活。 建议按照推荐的步骤完全去除磷酸酶。 d.反应体系中DNA的浓度过高会导致只产生线性DNA。建议连接体系中DNA的总浓度保持在1~10μg/ml的范围内。e.加入太多的连接产物转化到感受态细胞中会导致转化的失败。建议加入1~5μl的连接产物转化到50μl的感受态细胞中。f.含有PEG6000的情况下过长时间的连接，会逐渐产生抑制转化的大片段DNA，降低转化效率。g.连接产物在电穿孔前没有进行纯化。缓冲液中存在的盐和PEG6000均会抑制电穿孔实验。建议使用纯化柱对连接产物进行纯化，以尽可能去除缓冲液。h.空载体酶切不完全，会导致获得的克隆基本都是空载体而缺少含有插入片段的目的克隆。4.在解决转化效率问题时还应考虑哪些因素?a.感受态细胞不能存活或转化效率过低。建议使用新的感受态细胞。b.连接的DNA是否含有大肠杆菌拮抗的反向重复序列或串联重复序列。c.插入的DNA片段如果来自哺乳动物或者植物，可能含有能够被多种大肠杆菌株所降解的甲基化胞嘧啶。建议使用mcrA、mcrBC和mrr缺乏的大肠杆菌株。 d.构建的载体过大(＞10kb)，不能使用化学转化的方式，建议使用电穿孔转化方式。5.在限制性内切酶消化过程中存在哪些问题会导致T7 DNA Ligase的连接反应或后续的转化失败?a.酶切效率不高，没有实现完全酶切。如果切割发生在一个PCR片段的末端，需要确保有足够的酶切保护碱基，建议在酶切位点外侧额外增加6个碱基。并建议使用对照底物测试限制酶的活性。b.限制性内切酶没有完全失活。如果限制性内切酶不能热失活，可纯化DNA以尽可能地去除限制性内切酶。c.限制性内切酶切割DNA片段或载体时产生了星活性。建议凝胶电泳检测DNA，适当减少限制性内切酶的使用量或减少酶切反应时间。d.DNA或限制性内切酶中含有破坏DNA片段末端的核酸外切酶或磷酸酶时，建议纯化DNA。6.使用T7 DNA Ligase时应加入多少DNA?为促进环化DNA连接产物的形成，提高转化效率，加入的总DNA浓度应在1~10μg/ml之间，以实现有效连接。同时建议按照插入DNA片段与线性化载体摩尔比3:1的比例加入到反应体系中。摩尔比低于2:1会降低连接效率；摩尔比高于6:1会导致多个片段的插入。如果底物DNA的浓度无法确定，可尝试多种比例的连接。 参考文献：1.Aidan J. Doherty. J Biol Chem. 1996 May 10;271(19):11083-9.2.A J Doherty. Nucleic Acids Res. 1996 Jun 15;24(12):2281-7. |
| 英文描述 |
Aladdin's T7 DNA Ligase is a T7-derived, ATP-dependent dsDNA ligase recombinantly expressed in E. coli and purified using the PerfectProtein™ Technology Platform developed by aladdin. It ligates sticky ends more efficiently than blunt ends.Unlike T3 and T4 DNA Ligases, T7 DNA Ligase catalyzes the formation of a phosphodiester bond between 5' phosphate and 3' hydroxyl groups of dsDNA only, but does not efficiently ligates blunt-end dsDNA. Addition of PEG6000 over 20% to the reaction can improve the ligation of blunt-end dsDNA. However, under regular ligation conditions blunt-end DNA ligation does not occur. Therefore, T7 DNA Ligase is ideal for molecular biology applications in which both blunt and sticky ends of DNA are are present but only sticky ends needs to be joined [1,2].Please refer to Figure 1 for the performance of this product in ligating sticky ends of dsDNA.Figure 1. Ligation of sticky-end DNA fragments with T7 DNA Ligase from aladdin and Competitor. A. Colonies of DH5ɑ transformed with the ligation products; B. Colony PCR of clones randomly selected from plates in figure A. The experimental results demonstrates that this product has comparable performance with the widely received Competitor product in ligating sticky-end DNA. This figure is for reference only, which may vary due to different experimental conditions.s Application： Ligation of DNA fragments digested by restriction endonuclease, ligation of dsDNA and adapters, circularization of linear dsDNA, nick repair of dsDNA, site-specific mutagenesis, Golden Gate Assembly of DNA fragments, sticky-end specific ligation, etc.Source： Purified from E. coli with recombinant expression of DNA ligase from T7 phage.Definition of enzyme activity unit: One unit is defined as the amount of enzyme required for 50% ligation of 100ng HindIII fragments of λ DNA in a total reaction volume of 20μl in 30 minutes at 25°C in 1X T7 DNA Ligase Reaction Buffer.Purity: No DNA ligases other than T7 DNA Ligase, no DNA endonucleases and exonucleases, no RNase, no phosphodiesterase.Inactivation or inhibition： Ligation reactions without PEG6000 can be heat inactivated by incubation at 65℃ for 10 minutes.Precautions： ATP is a cofactor of T7 DNA Ligase, unlike E. coli DNA Ligase which uses NAD as a cofactor.T7 DNA Ligase does not effectively catalyse the ligation of blunt-end DNA fragments. For the ligation of blunt-end DNA, we recommend using the T4 DNA Ligase (, D7006).T7 DNA Ligase does not catalyze the ligation of ssDNA or RNA.The T7 DNA Ligase reaction contains 7.5% PEG6000. If the subsequent experiment is not compatible with PEG6000, a home-made ligation buffer without PEG6000 or the Ligation Buffer for T4 DNA Ligase (, D7006) can be used, but the activity of the T7 DNA Ligase will decrease by approximately 10-fold.We recommend using Pure™ Ultrapure Water (DNase/RNase-Free, Sterile) (, ST876) for the reaction.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. Set up the ligation reaction in a nuclease-free microfuge tube on ice as follows:ReagentVolume2X Reaction Buffer10μlVector DNAXμl (0.020pmol)Insert DNAYμl (0.060pmol)Ultrapure Water (9-X-Y)μl T7 DNA Ligase (3KU/μl)1μl Total Volume20μlNote 1: The molar ratio of insert DNA to linearrized vector should be 3:1.Note 2: T7 DNA Ligase should be added last.2. Mix well by pipetting and centrifuge briefly to collect the liquid to the bottom of the tube.3. Incubate at 25℃ (or room temperature) for 15-30 minutes.4. Chill on ice and transform 5μl of ligation product into 50μl of competent cells. The remaining sample can be stored at -20℃.Note 1: Heat deactivation cannot be performed, as it will dramatically reduce transformation efficiency.Note 2: To analyze the ligation efficiency, the reaction product can be subjected to agarose/PAGE analysis. If DNA recovery from agarose gel is required, we recommend using the DNA Gel Recovery Kit (, D0056).FAQ:1. What is the length of sticky ends that can be ligated by T7 DNA Ligase?T7 DNA Ligase can effectively catalyse the ligation of 2bp or longer sticky ends and cannot ligate 1bp sticky ends. The T7 DNA Ligase is unable to ligate blunt-end dsDNA under typical reaction conditions. However, when the reaction contains high concentration of PEG6000 (20-30% w/v), T7 DNA Ligase also has measurable ligation activity for blunt-end dsDNA.2. Can the T7 DNA Ligase be used with PEG6000-free buffers?Yes. If PEG6000 cannot be included in the reaction, a 2X Reaction Buffer without PEG6000 can be prepared.3. What are the potential factors that can cause transformation failure when using T7 DNA Ligase for ligation?a. Lack of ATP or Mg2+ in the reaction. ATP easily degrades upon repeated freeze-thaw of this product. We recommend storing this product in aliquot to avoid repeated freeze-thaw, or adding an appropriate amount of ATP in this product after being used for multiple times to ensure ligation efficiency.b. The presence of high salt or EDTA in the reaction. We recommend purifying the DNA substrate before ligation to remove the interference.c. Phosphatases such as CIP, BAP or SAP used for dephosphorylation of the DNA substrate are not completely inactivated before ligation. The phosphatases should be completely deactivated or removed according to the recommended procedures.d. Too high a concentration of DNA in the reaction can result in the production of linear DNA only. The total concentration of DNA in the ligation reaction should be within 1-10μg/ml.e. Adding too much ligation product to competent cells can cause failure of the transformation. We recommend transforming 1-5μl of ligation product to 50μl of competent cells.f. Prolonged ligation in the presence of PEG6000 can produce large DNA fragments that inhibit transformation.g. The ligation product was not purified prior to electroporation. The presence of both salt and PEG6000 has a significant impact on electroporation. We recommend purifying the ligation product using a purification column before electroporation.h. Incomplete digestion of the vector will result in lack of target clones containing the insert.4. What other factors could cause a low transformation efficiency?a. The competent cells have too low transformation efficiency. New batch of competent cells should be used.b. Whether the ligated DNA contains an E. coli antagonistic inverted repeat or a tandem repeat.c. The insert DNA of mammalian or plant origin may contain methylated cytosines that can be degraded by a wide range of E. coli strains. We recommend using mcrA, mcrBC and mrr deficient E. coli competent cells for transformation.d. The constructed plasmid that is ＞10kb should be transformed with electroporation, instead of chemical transformation.5. What problems in restriction endonuclease digestion can lead to failure of the ligation reaction or subsequent transformation?a. The digestion is not complete. If digestion occurs at the end of a PCR fragment, there must be approximately 6 protection bases at 5' end of the recognition site. Meanwhile, test the activity of the restriction enzyme with a control substrate.b. The restriction endonuclease is not completely inactivated. If the restriction endonuclease cannot be heat inactivated, the DNA can be purified with an appropriate method.c. Restriction endonuclease generated star activity. We recommend analyzing the cleaved DNA by gel electrophoresis, reducing the amount of restriction endonuclease, or reducing the digestion time appropriately.d. DNA purification is recommended when DNA or restriction endonucleases contain nucleic acid exonucleases or phosphatases that destroy the ends of DNA fragments.6. How much DNA should be added when using T7 DNA Ligase?To improve the formation of circular DNA as well as the transformation efficiency, the total DNA concentration in the ligation reaction should be within 1-10μg/ml to ensure effective ligation. The molar ratio of insert DNA to linearised vector is recommended to be 3:1. Molar ratios lower than 2:1 will reduce ligation efficiency, while molar ratios higher than 6:1 will result in the insertion of multiple fragments. If the concentration of substrate DNA cannot be determined, multiple ratios can be tested.References: 1. Aidan J. Doherty. J Biol Chem. 1996 May 10;271(19):11083-9.2. A J Doherty. Nucleic Acids Res. 1996 Jun 15;24(12):2281-7. |