Feb 10, 2022
Version 2
RNA Synthesis with Modified Nucleotides (E2050) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/2013/04/02/rna-synthesis-with-modified-nucleotides-e2050
Protocol Citation: New England Biolabs 2022. RNA Synthesis with Modified Nucleotides (E2050). protocols.io https://dx.doi.org/10.17504/protocols.io.bg54jy8wVersion 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 04, 2020
Last Modified: February 10, 2022
Protocol Integer ID: 37788
Keywords: synthesizing RNA with modified nucleotides, synthesizing biotin RNA, synthesizing dye-modified RNA
Abstract
This is the synthesis protocol for modified nucleotides using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050). The kit is capable of synthesizing biotin- or dye-modified RNA.
Guidelines
Figure 1 shows the time course of labeled RNA synthesis using 1 µg control template with Biotin-16-UTP and Fluorescein-12-UTP following the above reaction setup.
Modified ribonucleotides reduce transcription efficiency; therefore, lower transcription yields should be expected as compared to transcription using unmodified NTPs. In general, Biotin-NTP and Aminoallyl-NTP have an insignificant effect on yields, while lower yields can be expected for transcription reactions containing Fluorescein-NTP or Cy-NTP. In addition, transcripts containing modified ribonucleotides have reduced electrophoretic mobility due to higher molecular weight.
Figure 1. RNA synthesis with modified nucleotides
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
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
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.
The kit is capable of synthesizing biotin- or dye-modified RNA with the following protocol. The recommended molar ratio of modified NTP (Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP) to standard NTP is 1:2. The following reaction set-up assumes modified UTP is used. Please note that Dye- or Biotin-NTPs are not supplied with the kit.
Thaw the necessary kit components.
Mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes.
Assemble the reaction at Room temperature in the following order (Total reaction volume is 20 µL ):
| A | B | |
| Nuclease-free water | X µl | |
| NTP Buffer Mix | 5 µl (5 mM each NTP final) | |
| Modified UTP (10mM) | 5 µl (2.5 mM final) | |
| Template DNA | X µl (1 µg) | |
| T7 RNA Polymerase Mix | 2 µl | |
| Total reaction volume | 20 µl |
Note
Note that the ratio of modified nucleotide to standard nucleotide can be adjusted by varying the amount of the NTP Buffer Mix and modified nucleotide. For complete modified nucleotide substitution we recommended using the T7 High Yield RNA Synthesis Kit (NEB #E2040), in which the four nucleotides are supplied separately.
Mix thoroughly and pulse-spin.
Incubate at 37 °C for 02:00:00 .
Note
For short (< 300 nt) transcripts incubate at 37 °C for 04:00:00 –16:00:00 .
Note
Figure 1 shows the time course of labeled RNA synthesis using 1 µg control template with Biotin-16-UTP and Fluorescein-12-UTP following the above reaction setup.
Modified ribonucleotides reduce transcription efficiency; therefore, lower transcription yields should be expected as compared to transcription using unmodified NTPs. In general, Biotin-NTP and Aminoallyl-NTP have an insignificant effect on yields, while lower yields can be expected for transcription reactions containing Fluorescein-NTP or Cy-NTP. In addition, transcripts containing modified ribonucleotides have reduced electrophoretic mobility due to higher molecular weight.
Expected result
Figure 1. RNA synthesis with modified nucleotides
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 and 2 µL DNase I (RNase-free) , mix and incubate at 37 °C for 00:15:00 .
Proceed with purification synthesized RNA (we recommend the Monarch RNA Cleanup Kits, NEB #T2040 or #T2050) or analysis of transcription products by gel electrophoresis.