Standard RNA Synthesis (E2040)
Introduction
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.
Protocol
- 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:
REAGENT AMOUNT FINAL CONCENTRATION 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 DTT (0.1M)*
1 μl
5 mM
T7 RNA Polymerase Mix 2 μl
Total reaction volume 20 μl
*Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation.
- 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):
REAGENT AMOUNT FINAL CONCENTRATION Nuclease-free Water
X μl
10X Reaction Buffer
1.5 μl
0.75X final
NTP 1.5 μl each
7.5 mM each final
Template DNA
X μl
1 μg
DTT (0.1M)*
1 μl
5 mM
T7 RNA Polymerase Mix
1.5 μl
Total reaction volume
20 μl
*Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation.
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).