Home Protocols Capped RNA Synthesis (E2040)

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:

    REAGENT AMOUNT FINAL CONCENTRATION
    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
    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.

  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.

  7. Proceed with purification of synthesized RNA or analysis of transcription productss by gel electrophoresis (for purification, we recommend the Monarch RNA Cleanup Kits (NEB #T2040 or #T2050)).