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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
- 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:
|NTP Buffer Mix
||2 µl (2 mM each NTP final)
|Cap Analog (40 mM)
||4 µl (8 mM final)
||X µl (1 µg)
|T7 RNA Polymerase Mix
|Total reaction volume
- 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.
- 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 of synthesized RNA or analysis of transcription products by gel electrophoresis.