miRNA Cloning
RNA ligase uses ATP to adenylate the 5'-end of a single-stranded nucleic acid sequence. This activated adenylated oligomer is then covalently connected (ligated) to the 3'-OH of a second single-stranded sequence. Adenylated oligonucleotides containing a pyrophosphate linkage are substrates for T4 RNA Ligase in the absence of ATP1. Using the method of Unrau and Bartel2, IDT offers three different adenylated linker oligos that can be used in miRNA library construction.
Linker-1 is the original modban sequence employed by Lau and Bartel in 20013 and contains a Ban-I restriction site. Linker-2 contains Ava-I and Sty-I restriction sites. Linker-3 contains EcoR-I and Msp-I restriction sites and was adapted from Pfeffer and Tuschl4. All three linkers are modified with a 3’-terminal dideoxy-C (ddC) base to prevent self ligation. Experiments have shown miRNA linker performance can vary depending on the RNA source. For this reason, the miRNA Cloning Linker Pack – 3 Linker Types, 1 nmole each, provides small samples of each of the three linkers and is useful for optimizing methods before using precious RNA samples in large-scale library construction.
Linker oligonucleotides are provided lyophilized and are ready for use in cloning; just resuspend at the desired concentration and add to your ligation mix (using T4 RNA Ligase without ATP). Use of this reagent can improve cloning efficiency of miRNAs, which have a 5'-phosphate and will circularize if attachment of linkers is attempted using RNA Ligase in the presence of ATP.
Specifications

5' M.R.S (Multiple Restriction Site) miRNA Cloning Linker
The 5’ M.R.S Linker sequence is designed for use with any of the 3’ miRNA Cloning linkers. The sequence has been optimized for linking to the 5’ end of RNAs containing a 5’ phosphate group. The reaction is carried out with T4 RNA Ligase in the presence of 1 mm ATP. The sequence contains restriction endonuclease recognition sites compatible with Ban 1 (Linker 1), Sty I and Ava I (Linker 2) and EcoRI (Linker 3). Upon reverse transcription of doubly linked RNAs, the restriction endonuclease appropriate for the 3’ cloning linker will also generate compatible ends in the 5’ M.R.S Linker sequence permitting concatamerization and/or cloning. Further, the additional restriction sites in the M.R.S Linker, when matched with specific 3’ linkers can generate ends for directional cloning. For example, M.R.S Linker/Linker 3 digestion with Eco RI and Ban I will leave a Ban I 5’ end and an Eco RI 3’ end.

Name	1 nmole	5 nmole	Nmole/OD	MW	Sequence
M.R.S Linker	0.21 OD 7.0 μg	1.06 OD 35 μg	4.68	6989	5' TGGAATrUrCrUrCrGrGrGrCrArCrCrArArGrGrU 3 '

Technical Note
Instructions for Use
The miRNA cloning linkers are provided lyophilized and ready for use. IDT recommends that linkers be resuspended in nuclease-free water or IDTE Buffer (10mM Tris pH 8.0, 0.1mM EDTA) to a stock 100μM concentration (100pmole/μl). This is 10μl for the 1nmole product and 50μl for the 5nmole product. A typical RNA ligation reaction will require 1-5μl of linker. In addition, to avoid unwanted side reactions in the ligation, it is recommended that no more than 1-3 U of T4 RNA Ligase be used per reaction and that the ligation reaction not contain ATP.
References
1. Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase. England, T.E., Gumport, R.I., and Uhlenbeck, O.C. Proc Natl Acad Sci USA, 74:4839-42 (1977).
2. RNA-catalyzed nucleotide synthesis. Unrau, P.J., and Bartel, D.P. Nature, 395:260-63 (1998).
3. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. Science, 294:858-62 (2001).
4. Identification of microRNAs of the herpesvirus family. Pfeffer, S., Sewer, A., Lagos-Quintana, M., Sheridan, R., Sander, C., Grasser, F.A., van Dyk, L.F., Ho, C.K., Shuman, S., Chien, M. et al. Nat Methods, 2, 269-276 (2005).

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