Elsevier

Methods in Enzymology

Volume 317, 2000, Pages 109-123
Methods in Enzymology

[7] Joining of RNAs by splinted ligation

https://doi.org/10.1016/S0076-6879(00)17009-0Get rights and content

Publisher Summary

This chapter discusses the joining of RNAs by splinted ligation method. T4 DNA ligase is often the method of choice for joining two or more RNA molecules. The chapter also presents procedures for carrying out such ligation reactions. The first procedure gives generic conditions for RNA–RNA ligations using T4 DNA ligase. The two basic steps are hybridization of the RNA substrates to a bridging complementary (cDNA) oligonucleotide template and then incubation with T4 DNA ligase. This protocol is readily adaptable and can be used to make pico- to nano-molar amounts of linear ligated products. The second procedure gives a detailed protocol for the synthesis and confirmation of circular RNAs. The key for generating circles is to carry out the hybridization at low RNA and bridging cDNA oligonucleotide concentrations to favor the formation of circular monomers over linear dimers. The third and fourth procedures are for creating RNAs that contain a single internal radioactive label of high specific activity. The main differences between these protocols are the amounts of radioactivity used and whether the ligase reaction immediately follows the phosphorylation reaction without changing buffers and tubes.

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