Home Protocols Standard RNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080)

Standard RNA Synthesis Protocol using the HiScribe T7 mRNA Kit with CleanCap Reagent AG (NEB #E2080)

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 as needed.

  1. Thaw the necessary kit components to room temperature (Reaction Buffer, NTPs, CleanCap Reagent AG and Template DNA), mix and microfuge briefly to collect solutions to the bottom of the tubes.  Keep the Enzyme Mix on ice.

  2. If several reactions will be performed a master mix of Reaction Buffer, NTPs and CleanCap can be prepared according to the volumes per reaction below.  Use 12 µl of the Master Mix per reaction.

  3. Set up the following reaction at room temperature in the following order:

    REAGENTS AMOUNT FINAL CONCENTRATION
    Nuclease-free water X µl
    		  
    10X T7 CleanCap Reagent AG Reaction Buffer
    		 2 µl
    		  
    ATP (60 mM)
    		 2 µl
    		 6 mM final
    UTP (50 mM)
    		 2 µl
    		 5 mM final
    CTP (50 mM)
    		 2 µl
    		 5 mM final
    GTP (50 mM)
    		 2 µl
    		 5 mM final
    Cap Analog (40 mM)
    		 2 µl
    		 4 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.

  4. Gently mix the reaction by pipetting up and down and microfuge briefly.  Incubate at 37°C for 2 hours.

    For reaction times of 60 minutes or less, a water bath or heat block may be used.  For reactions longer than 60 minutes, we recommend using a dry air incubator or PCR instrument set to 37 C (with a heated lid) to prevent evaporation. For reactions with transcripts < 0.3 kb incubations up to 16 hours or overnight can be performed.

  5. Optional: Bring the reaction volume up to 50 µl with nuclease-free water.  Add 2 µl of DNase I, mix well and incubate at 37°C for 15 minutes.

  6. Proceed with mRNA purification.