5/26/2020 Typical Protocol for RNA Ligation (M0458) | NEB

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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:

COMPONENTS AMOUNT

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.

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