T3 DNA Ligase
规格
| 规格或纯度 | 不含除T3 DNA Ligase之外的其它种类的DNA连接酶，不含内切酶和外切酶，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T3 DNA Ligase之外的其它种类的DNA连接酶，不含内切酶和外切酶，不含RNA酶，不含磷酸酯酶。 |
| Cas编号 | |
| 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 1 minute at 25℃ in 1X T3 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T3 DNA Ligase，即T3 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T3噬菌体、ATP依赖型的双链DNA连接酶。T3 DNA Ligase能够有效催化粘性末端和平末端双链DNA分子的连接以及双链DNA或DNA与RNA杂合双链的缺刻修复。缺刻修复时，完整链为DNA，缺刻链5’端和3’端’分别为DNA-DNA、DNA-RNA、RNA-DNA或RNA-RNA都是可以被T3 DNA Ligase修复的，因此T3 DNA Ligase也可以用于生成DNA-RNA和RNA-DNA融合链接的核酸，以及用于基于DNA模板的RNA连接以形成较长的RNA片段。T3 DNA Ligase能够催化双链DNA中临近的5'磷酸和3'羟基之间形成磷酸二酯键，且对A/T突出末端的连接效率高于C/G末端。与T4 DNA Ligase相同，在T3 DNA Ligase反应体系中加入PEG6000可以显著提高其对平末端双链DNA的连接效率；而在缺少PEG6000的连接体系中，T3 DNA Ligase对平末端双链DNA分子的连接效率较低。另外，T3 DNA Ligase对NaCl的耐受性是T4 DNA Ligase的2倍，其在1.0M NaCl或KCl的条件下仍可保持95%的活性。因此，在涉及到高离子浓度连接的实验中，T3 DNA Ligase是理想的选择1,2]。阿拉丁生产的T3 DNA Ligase用于进行双链DNA粘性和平末端连接的效果参考图1。图1. 阿拉丁生产的T3 DNA Ligase 与N公司同类产品(Competitor)用于进行双链DNA粘性和平末端连接的效果图。图A为阿拉丁生产的或N公司(Competitor)的T3 DNA Ligase重组连接产物转化DH5α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HindIII酶切识别位点，另一端带有SmaⅠ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HindⅢ /SmaⅠ 对1kb的PCR产物进行双酶切，获得一端带有粘性末端，另一端带有平末端的双链线性DNA分子，作为T3 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，加入50ng经PCR扩增及HindⅢ/SmaⅠ双酶切产生的两端分别带有粘性末端和平末端的双链DNA片段，和50ng经HindⅢ/SmaⅠ双酶切线性化的pUC18载体(待插入DNA片段与pUC18载体的摩尔比为3:1)，再加入10µl的2X Reaction Buffer以及1μl的本产品或N公司(Competitor)的T3 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', "。图B为菌落PCR鉴定T3 DNA Ligase重组连接构建得到的克隆。实验结果表明，本产品与N公司的产品具有相当的连接粘性末端和平末端双链DNA的效果。菌落PCR鉴定使用的是pUC18载体的通用测序引物：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，PCR产物的克隆连接，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，高盐体系的连接，双链DNA缺刻修复，DNA引导的DNA与RNA连接，DNA引导的RNA连接。来源： 纯化自携带编码T3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T3 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。因此连接反应体系中一定要保持终浓度为1mM的ATP。本产品提供的2X Reaction Buffer中已经包含了ATP和连接粘末端所需的PEG6000。T3 DNA Ligase的催化底物是双链DNA或完整链为DNA的缺刻双链，不能直接用于单链DNA或RNA的连接反应，但可以用于DNA模板引导的单链DNA之间、单链RNA之间或单链DNA与单链RNA之间的连接。T3 DNA Ligase的反应体系中含有7.5% PEG6000。如果后续实验体系不兼容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，同时注意添加终浓度为1mM的ATP，但T3 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。在不含PEG的反应体系中，T3 DNA Ligase可进行热失活。如果反应体系中含有PEG6000，T3 DNA Ligase不能进行热失活，否则会显著降低后续的转化效率。如果连接体系中需要维持高浓度NaCl，建议使用不含PEG6000的缓冲液。在标准的载体与片段连接的20μl反应体系中，通常需要在25℃下反应30分钟。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 T3 DNA Ligase (3kU/μl) 1µl Total Volume 20µl 注1：建议按照插入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：T3 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.T3 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，推荐自行配制仅去除了PEG6000的2X Reaction Buffer。2.使用T3 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，会降低转化效率。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。3.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。4.在限制性内切酶消化过程中存在哪些问题会导致T3 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，可以纯化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。5.使用T3 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。6.T3 DNA连接酶对盐离子是否比T4 DNA连接酶具有更高的耐受性?是的。连接反应中T3 DNA Ligase能够耐受250-300mM的盐浓度。同时请注意，在PEG存在的连接体系中，DNA在高盐条件下会沉淀，并抑制连接反应的进行。7.T3 DNA Ligase能否被热失活?可以。在不含有PEG的反应体系中65℃孵育10分钟能够将T3 DNA Ligase热失活；但在含有PEG的反应体系中，T3 DNA Ligase可以进行热失活，但会影响后续的转化效率。因此在后续用于转化的情况下，不建议进行热失活。8.当使用T3 DNA连接酶时，应该使用什么对照来测试细胞和DNA?注：对每个对照均使用相同的DNA浓度，通常每次转化0.1-1.0ng的DNA。a.在感受态细胞中转化未经酶切线性化的载体并均匀涂布到不含有/含有抗生素的培养基平板上，可检测细胞活力和质粒的抗生素耐受性。b.检测线性化后的载体是否含有未被完全切割的质粒背景。将线性化后的载体转化感受态细胞，获得的克隆数目应小于步骤a中克隆数目的10%。c.使用切割并重新连接的质粒检测连接酶的活性和DNA末端的完整性。将切割并修饰后的载体转化感受态细胞，获得的克隆数目应接近步骤a中克隆数目。d.使用切割，磷酸化并重新连接的质粒检测由于磷酸化酶处理不完全所导致的背景。将切割，磷酸化并重新连接的质粒转化感受态细胞，获得的克隆数目应小于步骤a中克隆数目的10%。 参考文献：1.Cai L, Hu C, Shen S, Wang W, Huang W. 2004. 135(3):397-403.2.Lei Y, Washington J, Hili R. Org Biomol Chem. 2019. 17(7):1962-1965. |
| 英文描述 |
Aladdin's T3 DNA Ligase is a T3 phage-derived, ATP-dependent dsDNA ligase recombinantly expressed in E. coli and purified using the PerfectProtein™ Technology Platform developed by aladdin. It efficiently catalyzes the ligation of dsDNA molecules with either sticky or blunt ends, as well as the repair of nicks in dsDNA or DNA/RNA hybrids with an intact DNA strand. Therefore, T3 DNA Ligase can also can be used to generate DNA-RNA and RNA-DNA fusion products, as well as for RNA ligation to form longer RNA fragments, with intact DNA as template.T3 DNA Ligase is able to catalyse the formation of phosphodiester bonds between adjacent 5' phosphate and 3' hydroxyl groups in dsDNA and is more efficient at linking A/T protruding ends than C/G ends.Similar to T4 DNA Ligase, the addition of PEG6000 to the T3 DNA Ligase reaction significantly increases the ligation efficiency for blunt-ended dsDNA. The tolerance of T3 DNA Ligase to NaCl is twice that of T4 DNA Ligase, and it can maintain 95% activity under conditions of 1.0M NaCl or KCl. Therefore, T3 DNA Ligase is ideal choice for ligation under high ion concentrations [1,2].Please refer to Figure 1 for the performance of this product in ligating sticky and blunt ends of DNA.Figure 1. Sticky and blunt end ligation of dsDNA with T3 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 dsDNA with either sticky ends or blunt ends. This figur e is for reference only, which may vary due to different experimental conditions.s Application： Ligation of DNA fragments digested by restriction endonuclease, ligation of PCR products, ligation of dsDNA and adapters, circularization of linear dsDNA, nick repair of dsDNA, site-specific mutagenesis, ligation under high salt conditions, DNA-guided DNA-RNA or RNA ligation.Source： Purified from E. coli with recombinant expression of DNA ligase from T3 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 1 minute at 25℃ in 1X T3 DNA Ligase Reaction Buffer.Purity: No DNA ligases other than T3 DNA Ligase, no endonucleases and exonucleases, no RNase, no phosphodiesterase.Enzyme storage buffer： 10mM Tris-HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol (pH7.4 at 25℃).Inactivation or inhibition： Ligation reactions without PEG6000 can be heat inactivated by incubation at 65℃ for 10 minutes.Precautions： ATP is a cofactor of T3 DNA Ligase, unlike E. coli DNA Ligase which uses NAD as a cofactor. The 2X Reaction Buffer supplied with this product contains the ATP and the PEG6000 required for sticky end ligation.The catalytic substrate of T3 DNA Ligase is double-stranded DNA (dsDNA) or nicked DNA/RNA with an intact DNA strand. It cannot be used for ligation of single-stranded DNA or RNA, but can be used for DNA-guided ligation of single-stranded DNA, single-stranded RNA or between single-stranded DNA and single-stranded RNA.The T3 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, and ATP should be added at a final concentration of 1mM. However, the activity of the T3 DNA Ligase in the Ligase Buffer for T4 DNA Ligase will decrease by approximately 10-fold.In a reaction without PEG6000, T3 DNA Ligase can be heat inactivated. But the ligation reaction containing the PEG6000 cannot be heat inactivated. Otherwise the transformation efficiency will be significantly reduced.If a high concentration of NaCl is required for the ligation, we recommend using a buffer without PEG6000.A standard 20μl reaction typically requires 30 minutes at 25℃.We recommend using PurTM 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 T3 DNA Ligase (3kU/μl)1μl Total Volume20μlNote 1: The molar ratio of insert DNA to linearrized vector should be 3:1.Note 2: T3 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. Can the T3 DNA Ligase be used with PEG6000-free buffers?Yes. But the activity of T3 DNA ligase may be lower in such buffers.2. What are the potential factors that can cause transformation failure when using T3 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.3. 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. 4. 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.5. How much DNA should be added when using T3 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 cause a reduction of 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.6. Is T3 DNA ligase more tolerant to salt ions than T4 DNA ligase?Yes. The T3 DNA Ligase is able to tolerate 250-300mM of salt. Please also note that in the presence of PEG, DNA may precipitate in high salt conditions, which can inhibit the ligation reaction.7. Can T3 DNA Ligase be heat inactivated?Yes. T3 DNA Ligase can be heat inactivated by incubation at 65℃ for 10 minutes in a reaction without PEG. However, do not heat inactivate the ligation reaction containing PEG, as it will dramatically reduce transformation efficiency. 8. What control should be used to test cells and DNA when using T3 DNA ligase?Note: Use the same DNA concentration for each control and 0.1-1.0ng of DNA for each transformation.a. Cell viability and antibiotic resistance can be assayed by transforming the undigested vector into competent cells which are then spread evenly onto plates with/without antibiotics.b. Detect whether the vector is linearised completely. The number of clones obtained from linearised vector transformation should be less than 10% of the clone number in step a.c. Use re-ligated plasmids to test ligase activity and DNA end integrity. Transform digested and modified vectors into competent cells and the number of clones obtained should be close to the number of clones in step a.d. Use the digested, phosphorylated and re-ligated plasmids to detect background caused by incomplete dephosphorylatiion. Transformation of the cleaved, phosphorylated and re-ligated plasmid into competent cells should result in less than 10% of clones from step a.References: 1. Cai L, Hu C, Shen S, Wang W, Huang W. 2004. 135(3):397-403.2. Lei Y, Washington J, Hili R. Org Biomol Chem. 2019. 17(7):1962-1965. |