Feb 10, 2022
Version 2
Standard 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. Standard RNA Synthesis (E2050). protocols.io https://dx.doi.org/10.17504/protocols.io.bg55jy86Version 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: June 03, 2020
Last Modified: February 10, 2022
Protocol Integer ID: 37789
Keywords: RNA Synthesis,
Abstract
This is the synthesis protocol using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050)
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.
Reaction time depends on template amount, quality and RNA transcript length. For reactions with transcripts longer than 0.3 kb, 2 hour incubation should give you the maximum yield. For reaction times of 60 minutes or less, a water bath or heating block may be used; for reaction times longer than 60 minutes, we recommend using a dry air incubator or a thermocycler to prevent evaporation of the sample.
For reactions with short RNA transcripts (< 0.3 kb), we recommend an incubation time of 4 hours or longer. It is safe to incubate the reaction for 16 hours (overnight). For example, we have achieved good yield with only 0.2 μg plasmid template encoding a 50-mer RNA by incubating the reaction overnight at 37°C.
Reaction set up for short transcripts (< 0.3 kb):
| A | B | |
| Nuclease-free water | X µl | |
| NTP Buffer Mix | 10 µl (6.7 mM each NTP final) | |
| Template DNA | X µl (1 µg) | |
| T7 RNA Polymerase Mix | 2 µl | |
| Total reaction volume | 30 µl |
Compared to the standard reaction, this reaction uses 10 μl more water. The volume of NTP Buffer Mix and T7 RNA Polymerase Mix, however, remains the same. The kit contains sufficient materials for 50 reactions.
Note that the amount of NTP Buffer Mix in a standard 20 μl reaction can vary from 2 to 10 μl. The final yield is proportional to the amount of input nucleotides, meaning that the nucleotide incorporation efficiency remains the same when different amounts of NTP are used. Figure 1 shows the time course of standard RNA synthesis from 1 μg control DNA template coding for a 1.8 kb RNA transcript with the HiScribe T7 Quick Kit using 10 μl and 5 μl NTP Buffer Mix in a 20 μl reaction.
Figure 1. RNA synthesis with different amounts of NTP.
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
HiScribe T7 High Yield RNA Synthesis Kit - 50 rxnsNew England BiolabsCatalog #E2040S
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
We strongly recommend wearing gloves.
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.
Thaw the necessary kit components.
Mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Keep On ice .
Assemble the reaction at Room temperature in the following order:
| A | B | |
| Nuclease-free water | X µl | |
| NTP Buffer Mix | 10 µl (10 mM each NTP final) | |
| Template DNA | X µl (1 µg) | |
| T7 RNA Polymerase Mix | 2 µl | |
| Total reaction volume | 20 µl |
Mix thoroughly and pulse-spin in a microfuge.
Incubate at 37 °C for 02:00:00 .
Note
Reaction time depends on template amount, quality and RNA transcript length. For reactions with transcripts longer than 0.3 kb, 2 hour incubation should give you the maximum yield. For reaction times of 60 minutes or less, a water bath or heating block may be used; for reaction times longer than 60 minutes, we recommend using a dry air incubator or a thermocycler to prevent evaporation of the sample.
For reactions with short RNA transcripts (< 0.3 kb), we recommend an incubation time of 4 hours or longer. It is safe to incubate the reaction for 16 hours (overnight). For example, we have achieved good yield with only 0.2 μg plasmid template encoding a 50-mer RNA by incubating the reaction overnight at 37°C.
Reaction set up for short transcripts (< 0.3 kb):
| A | B | |
| Nuclease-free water | X µl | |
| NTP Buffer Mix | 10 µl (6.7 mM each NTP final) | |
| Template DNA | X µl (1 µg) | |
| T7 RNA Polymerase Mix | 2 µl | |
| Total reaction volume | 30 µl |
Compared to the standard reaction, this reaction uses 10 μl more water. The volume of NTP Buffer Mix and T7 RNA Polymerase Mix, however, remains the same. The kit contains sufficient materials for 50 reactions.
Note that the amount of NTP Buffer Mix in a standard 20 μl reaction can vary from 2 to 10 μl. The final yield is proportional to the amount of input nucleotides, meaning that the nucleotide incorporation efficiency remains the same when different amounts of NTP are used. Figure 1 shows the time course of standard RNA synthesis from 1 μg control DNA template coding for a 1.8 kb RNA transcript with the HiScribe T7 Quick Kit using 10 μl and 5 μl NTP Buffer Mix in a 20 μl reaction.
Expected result
Figure 1. RNA synthesis with different amounts of NTP.
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 30 µL nuclease-free water to each 20 μl reaction, followed by 2 µL DNase I (RNase-free) , mix and incubate at 37 °C for 00:15:00 .
Note
Standard reactions are capable of generating large amounts of RNA, at concentrations up to 10 mg/ml. As a result, the reaction mixture is quite viscous. It is easier to perform DNase treatment after the reaction mixture is diluted.
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.