Universal miRNA Cloning Linker (5′ adenylated, 3′ blocked) (C007B-566615)

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 isApplication：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.