Standard RNA Synthesis (E2040)
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 bottom of tubes. Keep on ice.
- If you are planning to run many reactions, it is convenient to prepare a master mix by combining equal volumes of the 10X reaction buffer and four ribonucleotide (NTP) solutions. Use 10 μl per reaction.
- Assemble the reaction at room temperature in the following order:
Nuclease-free water X μl 10X Reaction Buffer 2 μl ATP (100 mM) 2 μl 10 mM final GTP (100 mM) 2 μl 10 mM final UTP (100 mM) 2 μl 10 mM final CTP (100 mM) 2 μl 10 mM final Template DNA X μl 1 μg T7 RNA Polymerase Mix 2 μl Total reaction volume 20 μl
- Mix thoroughly, pulse-spin in microfuge. Incubate at 37°C for 2 hours. The yield will not be compromised if the incubation temperature is within the range of 35–40°C.
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 PCR instrument, to prevent evaporation.Figure 2 shows the time course of standard RNA synthesis from 1 μg linearized plasmid DNA tem- plates coding for 0.3 kb, 0.8 kb and 1.8 kb RNA transcripts with the HiScribe T7 High Yield RNA Synthesis Kit. For reactions with transcripts longer than 0.3 kb, 2 hour incubation should give you the maximum yield. Figure 3 shows DNA template titrations for 0.3 kb and 1.8 kb RNA transcripts with the HiScribe T7 High Yield RNA Synthesis Kit at 37°C for 2 hours.
Figure 3 on the main product page shows DNA template titrations for 0.3 kb and 1.8 kb RNA transcripts with the HiScribe T7 High Yield RNA Synthesis Kit at 37°C for 2 hours. For reactions with short transcripts (< 0.3 kb), increase template DNA up to 2 μg or incubate for longer time to achieve maximum yield.
For reactions with short RNA transcripts (< 0.3 kb), follow the reaction set up below and incubate the reaction for 4 hours or longer. It is safe to incubate the reaction for 16 hours (overnight).
Reaction set up for short transcripts (< 0.3 kb):
10X Reaction Buffer
NTP 1.5 μl each
7.5 mM each final
T7 RNA Polymerase Mix
Total reaction volume
With this set up, the kit contains sufficient materials for 65 reactions.
- Optional: DNase treatment to remove DNA template. Standard reactions normally generate 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. To remove template DNA, add 70 μl nuclease-free water, 10 μl of 10X DNase I Buffer, and 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.
- Proceed with purification of synthesized RNA or analysis of transcription products by gel electrophoresis (for purification, we recommend the
Monarch RNA Cleanup Kits (NEB #T2040 or #T2050).