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  • Capped RNA Synthesis (E2050)

    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.

    1. 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.

    2. Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to the bottoms of tubes.

    3. Assemble the reaction at room temperature in the following order:
      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 (1 µg)
      T7 RNA Polymerase Mix 2 µl
      Total reaction volume 20 µl 

    4. 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.
    5. Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.

    6. Proceed with purification of synthesized RNA or analysis of transcription products by gel electrophoresis.