Typical Protocol for RNA Ligation (M0458)

1.    Prepare 1 mM GTP by diluting the 10 mM stock in nuclease-free water. Store at –20°C for repeated use.

2.    Assemble the following reaction in a nuclease-free PCR tube on ice:
 3´-phosphate RNA donor
 10 pmol (0.5 pmol/µl)
 5´-OH RNA acceptor
 10 pmol (0.5 pmol/µl)
 RtcB Reaction Buffer (10X)
 2 µl
 1 mM GTP
 2 µl
 10 mM MnCl2
 2 µl
 RtcB RNA Ligase
 1 µl (15 pmol)
 Nuclease-free Water
 Up to 20 µl

3.    Incubate at 37°C for 1 hour.

4.    We recommend cleaning up your reactions before moving on to downstream applications. This can be achieved by using a spin column-based method or phenol:chloroform extraction followed by ethanol precipitation.