RNA Synthesis with Modified Nucleotides (E2050)

New England Biolabs

Published: 2022-02-10 DOI: 10.17504/protocols.io.bg54jy8w

synthesizing RNA with modified nucleotides

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.

Before start

We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20µL 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.

Steps

1.

Thaw the necessary kit components.

2.

Mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes.

3.

Assemble the reaction at Room temperature in the following order (Total reaction volume is 20µL):

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 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.), in which the four nucleotides are supplied separately.

4.

Mix thoroughly and pulse-spin.

5.

Incubate at 37°C for 2h 0m 0s.

Note

For short (< 300 nt) transcripts incubate at 37°C for 4h 0m 0s–16h 0m 0s.

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.

Citation

Figure 1. RNA synthesis with modified nucleotidesReactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.

6.

Optional step : To remove template DNA, add 30µL and 2µL, mix and incubate at 37°C for 0h 15m 0s.

7.

Proceed with purification synthesized RNA (we recommend the Monarch RNA Cleanup Kits, NEB #T2040 or #T2050) or analysis of transcription products by gel electrophoresis.

RNA Synthesis with Modified Nucleotides (E2050)

Abstract

Before start

Steps

推荐阅读