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 formulation allows for efficient capped RNA synthesis using cap analog (ARCA). The recommended ratio of cap analog to GTP is 4:1. Increasing the ratio of cap analog to GTP will increase the proportion of capped RNA transcripts; however, it also significantly decreases the yield of the reaction. Cap analogs are sold separately. Please refer to the companion products section.
Prepare 40 mM cap analog. Cap analog (ARCA, NEB #S1411) is supplied in a lyophilized form of 1 µmol per tube. Dissolving it in 25 μl nuclease-free water will yield a concentration of 40 mM.
Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to the bottoms of tubes.
Assemble the reaction at room temperature in the following order:
REAGENT
AMOUNT
FINAL CONCENTRATION
Nuclease-free water
X µl
NTP Buffer Mix
2 µl
2 mM each NTP final
Cap Analog (40 mM)
4 µl
8 mM final
Template DNA
X µl
1µg
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 and incubate at 37°C for 2 hours. The yield per reaction is 30–40 μg RNA with approximately 80% capped RNA transcripts. Figure 1 shows the time course of capped RNA synthesis from 1 µg control template. Most reactions will be complete in 1 hour.
Figure 1. Capped RNA Synthesis with ARCA Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop Spectrophotometer.
Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.
Proceed with purification synthesized RNA (we recommend the Monarch RNA Cleanup Kits, NEB #T2040 or #T2050) or analysis of transcription products by gel electrophoresis.
