Home Protocols Standard mRNA Synthesis (E2065)

Standard mRNA Synthesis (E2065)

Please wear gloves when setting up RNA transcription reactions. Be sure to use nuclease-free tubes and reagents to avoid RNase contamination. Transcription 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.

Standard mRNA Synthesis

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

    Assemble the reaction at room temperature in the following order:
    Nuclease-free water to 20 µl  
    2X ARCA/NTP Mix 10 µl 1 mM GTP, 4 mM ARCA,
    1.25 mM CTP, 1.25 mM
    UTP, >1.25 mM ATP final
    Template DNA X µl 1 µg
    T7 RNA Polymerase Mix 2 µl  
    Total reaction volume 20 µl  
  2. Mix thoroughly and pulse-spin in a microfuge. Incubate at 37°C for 30 minutes.

    Do not heat the reaction. Do not purify the RNA. Proceed to DNase treatment step or store the reaction at -20°C for a few days.

    Reaction time depends on template amount, quality and RNA transcript length. For reactions with transcripts longer than 0.5 kb, 30 min incubation should give you the maximum yield.

    For reactions with short RNA transcripts (< 0.5 kb), incubation time of 1 hour or longer is necessary to achieve good yield. It is safe to incubate the reaction for 16 hours (overnight).

    For reaction times of 60 minutes or less, a water bath or heating block may be used; for reaction times longer than 60 minutes, please use a dry air incubator or PCR machine.

  3. DNase treatment to remove template DNA. Add 2 μl of DNase I, mix well and incubate at 37°C for 15 minutes.

    DNase treatment is optional if the template does not interfere with downstream experiment. If left untreated, DNA template containing eukaryotic promoters may produce a background in mRNA transfection experiments.

  4. Save 1 μl for gel analysis if desired. Do not heat the reaction or purify the RNA. Proceed to tailing reaction. For purification, we recommend the Monarch RNA Cleanup Kits (NEB #T2040 or #T2050).