Universal miRNA Cloning Linker (5′ adenylated, 3′ blocked)

阿拉丁生产的Universal miRNA Cloning Linker (5´腺苷化3´封闭)，即通用miRNA克隆接头(5´腺苷化3´封闭)或Universal miRNA Cloning Linker (5'adenylated, 3'blocked)，是一种常用于miRNA等3'端为羟基的RNA或3'端为羟基的单链DNA在克隆、高通量测序建库或PCR检测等时，在3'端添加的接头。本产品5´端腺苷酰化(也称5´端预腺苷酰化或5´端预腺苷化，5′ pre-adenylated或5′ pre-adenylation)，3´端氨基封闭，可以用于3'端为羟基的RNA的3'端接头的添加。RNA连接酶可以识别“活化”的5´端腺苷酰化的本产品，在无需ATP存在的条件下，可将本产品的5´端与另外一条单链寡核苷酸的3´端羟基共价连接。利用5´端腺苷酰化，3´端封闭的本产品，加入RNA连接酶，在无需添加ATP的条件下，就能实现本产品5'端和目标序列3'羟基的特异性共价连接。例如和miRNA连接时，可以有效避免miRNA的自连、不同miRNA之间的连接、本产品的自连或本产品不同链之间的连接，而仅发生本产品5'端和miRNA 3'羟基之间的特异性连接。本产品的电泳图和连接效果图请参考图1。图中可见，本产品条带清晰，纯度高，与5'端为磷酸化的序列相比有显著的电泳迁移率差异(图1A)；并且本产品使用"T4 RNA ligase 2 , truncated与ssRNA连接时，连接效率非常高(图1B)。图1. Universal miRNA Cloning Linker Linker (5´腺苷化3´封闭)及其连接效果图图。图A. 本产品5´-rAppCTGTAGGCACCATCAAT–NH2-3´ (AppDNA)与5´-pCTGTAGGCACCATCAAT–NH2-3´ (pDNA)的电泳对比图。图B. 本产品(AppDNA)与ssRNA使用T4 RNA ligase 2 , truncated的连接效果图。图中可见本产品与ssRNA有非常高的连接效率。本产品的寡核苷酸序列是：5´-rAppCTGTAGGCACCATCAAT–NH2-3´。本产品的序列同NEB公司的S1315S Universal miRNA Cloning Linker。本产品以水溶液的形式存在，浓度为40ng/µl，即6.9µM。 用途 ： 本产品可以应用于小RNA克隆、miRNA文库构建。本产品的1µg小包装约通常可以进行8-17次连接反应，5µg中包装约通常可以进行40-85次连接反应。具体的连接反应次数和反应体系的体积和本产品的用量有关。 注意事项 ： 本产品4ºC保存时应避免靠近冰箱壁，以免局部温度过低导致沉淀析出。如果有沉淀析出，室温摇晃片刻，再放置在4ºC冰箱过夜或更长时间，待沉淀消失后再使用。本产品一般适用于293细胞悬浮培养，不推荐用于没有经驯化过的贴壁293细胞的培养。使用本产品应注意无菌操作，避免污染。一般不建议补充抗生素，以免影响细胞的正常生长速度，但如果不能再高洁净度细胞培养室内操作，仍需加入适量的抗生素。若使用过程中发现培养液浑浊、沉淀等异常现象，则不能继续使用。本产品中已添加的L-谷氨酰胺(约6mM)是细胞培养所必需的营养成分，一般无需额外再添加，否则可能产生细胞毒性。但L-谷氨酰胺在溶液中不太稳定、易分解，若本培养液长期放置后再使用，发现细胞生长变慢，可在使用前加入适量的L-Glutamine (100X) 或可以稳定保存的L-Ala-Gln (100X) ，以维持细胞的正常生长代谢。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.使用本产品与ssRNA的3'羟基末端连接推荐使用T4 TNA Ligase 2, truncated。也可以使用T4 TNA Ligase 2, truncated,K227Q，或T4 TNA Ligase 2, truncated,T4 TNA Ligase 2, truncatedKQ 。2.参考下表在冰浴中配制如下反应体系：ReagentVolumeFinal ConcentrationssRNAxµl0.5µMDEPC-treated Water(1.1 or 2.55)-xµl-Universal miRNA Cloning Linker (6.9pmol/µl)2.9 or 1.45µl1 or 0.5µMPEG8000 (50%, RNase Free)12µl0.310X Reaction Buffer2µl1 XRNase Inhibitor (40U/μl)1µl2U/μlT4 RNA Ligase2, truncated (200U/µl)1µl10U/µl -Total Volume20µl-注意：a.由于涉及RNA操作，需要严格按照RNA操作的规范进行，避免RNase污染，相关试剂和耗材需要经过DECP处理去除RNase或者确保是RNase free的。推荐在连接体系中添加RNase Inhibitor，以避免ssRNA或其连接产物的降解，尽管经测试我们发现在严格操作的情况下，不加RNase Inhibitor也不会导致ssRNA或其连接产物发生明显降解。b.进行连接反应时，如果样品RNA比较珍贵，希望充分被连接，可以按照接头和样品RNA的摩尔比为2:1的比例进行连接；如果样品RNA比较充裕，同时感觉本产品的成本偏高时，可以按照接头和样品RNA的摩尔比摩尔比1:1甚至1:2的比例进行连接反应，这样可以很好地节省本产品。c.如果同时进行多个连接反应，可以把上表中除ssRNA之外的所有溶液和酶提前预混合，然后再分装到各反应管内。3.连接反应：25℃, 孵育60min。为了使连接反应更加充分，可以适当延长连接反应时间。4.终止反应：65℃，孵育15min。 Aladdin's Universal miRNA Cloning Linker (5´ adenylated, 3´ blocked) can be covalently ligated to the 3' OH of RNA or single-stranded DNA for cloning, NGS library preparation, and PCR assays, etc.RNA ligase recognizes the 5' adenylated oligo and ligates its 5' end to the 3´ OH of a second single-stranded oligonucleotide in the absence of ATP. In a mixture of nucleic acids, use of this product with RNA ligase results in ligation of the target oligo only, which does not require ATP.Please refer to Figure 1 for the electrophoresis analysis and ligation effect of this product. As shown in the figure, this product show a clear band with high purity. Its migration rate (AppDNA) is obviously different from 5' phosphorylated sequence (pDNA) (Figure 1A). The ligation efficiency is also very high when using the T4 RNA ligase 2 , truncated to ligate this product with ssRNA (Figure 1B).Figure 1. The electrophoresis analysis and ligation effect of Aladdin's Universal miRNA Cloning Linker Linker (5´ adenylated, 3´ blocked) . A. Migration rate of 5´-rAppCTGTAGGCACCATCAAT-NH2-3´ (AppDNA) and 5´-pCTGTAGGCACCATCAAT-NH2-3´ (pDNA); B. Ligation of this product (AppDNA) with ssRNA in the presence of T4 RNA ligase 2 , truncated.The oligonucleotide sequence of this product is Application ： This product can be used for short RNA cloning and miRNA library preparation.R0702S provides sufficient linker for 8-17 ligation reactions and R0702M is sufficient for 40-85 ligation reactions. The specific number of reactions depends on the reaction volume and the amount of linker used.Packing List：ItemComponentQuantityR0702SUniversal miRNA Cloning Linker (40ng/µl)1µgManual-1 copyItemComponentQuantityR0702MUniversal miRNA Cloning Linker (40ng/µl)5µgManual-1 copyStorage Conditions：Store at -20℃ for up to one year, or -80℃ for longer.Precautions：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. Precautions ： 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. T4 TNA Ligase 2, truncated is recommended for the ligation reaction. T4 TNA Ligase 2, truncated, K227Q or T4 TNA Ligase 2, truncated, KQ can also be used. 2. Prepare the following reaction on ice.ReagentVolumeFinal ConcentrationssRNAxµl0.5µMDEPC-treated Water(1.1 or 2.55)-xµl-Universal miRNA Cloning Linker (6.9pmol/µl)2.9 or 1.45µl1 or 0.5µMPEG8000 (50%, RNase Free)12µl30%10X Reaction Buffer2µl1 X RNase Inhibitor (40U/μl)1µl2U/μlT4 RNA Ligase2, truncated (200U/µl)1µl10U/µl -Total Volume20µl-Note:a. As the reaction is RNA involved, the reagents and consumables should be RNase free. To avoid degradation of ssRNA or its ligation products, the addition of RNase Inhibitor (, R0102) is recommended. b. If the sample RNA is precious and sufficient ligation is desired, the molar ratio of the linker and the sample RNA can be set to 2:1. If the sample RNA is abundant and the linker needs to be saved, the molar ratio of the linker and the sample RNA can be set to 1:1 or even 1:2. c. When multiple reactions are required, prepare a master mix including all reagents except the substrate RNA and then dispense to different nuclease-free tubes. Finally, add the substrate RNA into each tube.3. Incubate at 25℃ for 60min or longer for more sufficient ligation.4. Terminate the reaction by incubating at 65℃ for 15min.

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