Universal miRNA Cloning Linker
- Can be used for the cloning of short RNAs according to method described below
- Sequence: 5′ rAppCTGTAGGCACCATCAAT–NH2 3′
This 5´ adenylated, 3´ blocked oligodeoxynucleotide can be used for cloning short RNAs according to the procedure of Bartel (1). RNA ligase recognizes the "activated" adenylated oligo and covalently ligates its 5´ end to the 3´ OH of a second single stranded sequence in the absence of ATP. In a mixture of nucleic acids use of the 5´ adenylated, 3´ blocked oligo with RNA ligase (w/o ATP) results in ligation of the target oligo only.
Linker/RNA Ligation Protocol: The Universal miRNA cloning linker has a 3´ blocking group (amine) to prevent self ligation, circularization and ligation to RNA at the 5´ end. It also contains the BanI restriction site compatible with the miRNA cloning protocol used by Bartel (1).
For ligation, resuspend the lyophilized oligonucleotide in RNAse-free water to 100 μM stock concentration. (A resuspension volume of 8.6 μl will give a 0.58 μg/μl or 100 μM stock solution). Ligations are carried out with T4 RNA Ligase I in the absence of ATP. Approximately 1 μg is typically used per reaction. See reference 1 for a detailed protocol.
Sequence5´ rAppCTGTAGGCACCATCAAT–NH2 3´
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