Feb 14, 2022
Version 2
Capped RNA Synthesis (E2050) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2022. Capped RNA Synthesis (E2050). protocols.io https://dx.doi.org/10.17504/protocols.io.bddgi23wVersion created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 07, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 33928
Keywords: Capping RNA, capped RNA using cap analog, capped RNA syntehsis with ARCA, E2050
Abstract
The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA).
Guidelines
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.
Cap analog (ARCA, NEB #S1411) is supplied in a lyophilized form of 1 µmol per tube. Dissolving it in 25 μl nuclease-free water will yield a concentration of 40 mM.
The yield per reaction is 30–40 μg RNA with approximately 80% capped RNA transcripts. Figure 1 shows the time course of capped RNA synthesis from 1 µg control template. Most reactions will be complete in 1 hour.
Figure 1. Capped RNA Synthesis with ARCA
Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.
Materials
MATERIALS
HiScribe T7 Quick High Yield RNA Synthesis Kit - 50 rxnsNew England BiolabsCatalog #E2050S
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
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.
Prepare 40 millimolar (mM) cap analog .
Note
Cap analog (ARCA, NEB #S1411) is supplied in a lyophilized form of 1 µmol per tube. Dissolving it in 25 μl nuclease-free water will yield a concentration of 40 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 02:00:00 .
Note
The yield per reaction is 30–40 μg RNA with approximately 80% capped RNA transcripts.Figure 1 shows the time course of capped RNA synthesis from 1 µg control template. Most reactions will be complete in 1 hour.
Figure 1. Capped RNA Synthesis with ARCA. Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.
Optional step: To remove template DNA, add 2 µL DNase I (RNase-free) , mix and incubate at 37 °C for 00:15:00 .
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.