mRNA Synthesis with Modified Nucleotides (E2060)
The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP.
- Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order:
REAGENT AMOUNT FINAL CONCENTRATION Nuclease-free water to 20 µl 2X ARCA/NTP Mix 10 µl 1 mM GTP, 4 mM ARCA,
1.25 mM CTP, 1.25 mM
UTP, >1.25 mM ATP final
5mCTP, 10 mM 2.5 µl 1.25 mM 5mCTP final Pseudo-UTP, 10 mM 2.5 µl 1.25 mM Pseudo-UTP final Template DNA X µl 1 μg DTT (0.1)*
T7 RNA Polymerase Mix 2 µl Total reaction volume 20 µl
*Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation.
- Follow standard mRNA synthesis protocol.