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  • Capped RNA Synthesis (E2040)

    Introduction

    The recommended ratio of cap analog to GTP is 4:1. Cap analogs are sold separately. Please refer to the ordering information section for more information.

    Protocol

    1. Thaw the necessary kit components, mix and pulse-spin in a microfuge to collect solutions to bottom of tubes. Keep on ice.

    2. Make 20 mM GTP solution by combining 2 μl of 100 mM GTP and 8 μl of nuclease-free water. Extra 20 mM GTP can be stored at -20°C for future use.

    3. Prepare cap analog at the concentration of 40 mM.

    4. 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
      UTP (100 mM) 2 μl 10 mM final
      CTP (100 mM) 2 μl 10 mM final
      GTP (20 mM) 2 μl 2 mM 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  
    5. Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours.

      The yield per reaction is 40–50 μg RNA with approximately 80% capped RNA transcripts. Table 1 shows the effect of varying the ratio of cap analog to GTP on the yield of RNA. 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 transcription reaction. A ratio of cap analog to GTP of 4:1 is preferably used.

      Table 1. Effect of cap analog:GTP ratios on RNA yield
      Cap Analog: GTP Ratio Concentration of Cap
      Analog: GTP (mM)
      RNA Yield (μg) in
      2 hours
      % Capped RNA
      0:1 0:10 180 0
      1:1 5:5 90–120 50
      2:1 6.7:3.3 60–90 67
      4:1 8:2 40–50 80
      8:1 8.9:1.1 20–25 89
    6. Optional: To remove template DNA, add 2 μl of DNase I (RNase-free), mix and incubate at 37°C for 15 minutes.