Capped RNA Synthesis (E2050)
New England Biolabs
Abstract
The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA).
Before start
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA). The recommended ratio of cap analog to GTP is 4:1. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts; however, it also significantly decreases the yield of the reaction. Cap analogs are sold separately. Please refer to the companion products section.
Steps
Prepare 40millimolar (mM).
Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to the bottoms of tubes.
Assemble the reaction at Room temperature in the following order:
| A | B |
|---|---|
| Reagent | Volume |
| Nuclease-free water | X µl |
| NTP Buffer Mix | 2 µl (2 mM each NTP final) |
| Cap Analog (40 mM) | 4 µl (8 mM final) |
| Template DNA | X µl (1 µg) |
| T7 RNA Polymerase Mix | 2 µl |
| Total reaction volume | 20 µl |
Mix thoroughly and pulse-spin.
Incubate at 37°C for 2h 0m 0s.
Optional step : To remove template DNA, add 2µL, mix and incubate at 37°C for 0h 15m 0s.
Proceed with purification of synthesized RNA (we recommend the Monarch RNA Cleanup Kits, NEB #T2040 or #T2050) or analysis of transcription products by gel electrophoresis.
