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Star Republic: Guide for Biologists

Polyadnylation --- Ambion PolyA tailing

This protocol uses Ambion's PolyA tailing kit.

1. Start with completed, DNase-treated in vitro transscription raction. Don't remove unincorporated nucleotides or add EDTA.

2. Make the tailing reaction mix in the following order:

in vitro transctiption RNA 20 ml
Nuclease-free ddH2O 36 ml
5X E-PAP Buffer 20 ml
25 mM MnCl2 10 ml
10 mM ATP 10 ml

3. Mix and remove 0.5 ml as negative control.

4. Add 4 ml E-PAP enzyme.

5. Mix and incubate at 37 C for 1 hour.

6. Put on ice or store at -20 C.