|
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.
| 