Protocol for Co-transcriptional capping using CleanCap® Reagent AG from TriLink and HiScribe T7 High Yield RNA Synthesis Kit from New England Biolabs®


Reagents required:

1.  NEB HiScribe T7 High Yield RNA Synthesis Kit, NEB #E2040S
2.  TriLink CleanCap® Reagent AG, N-7113, or CleanCap® Reagent AG (3’ OMe), N-7413

Reagents not supplied:

1.  Double-stranded DNA template

**NOTE: CleanCap AG requires modification to the sequence just downstream of the T7 promoter sequence, replacing the “GG” that follows the T7 promoter sequence with an “AG” as follows:

Standard T7 promoter (underlined) with GG:


Sequence required for CleanCap® with AG:


2.  NEB Monarch® RNA Cleanup Kit (500 µg), T2050

Co-transcriptional Capping Protocol:
(NOTE: 5 mM final each NTP instead of 10 mM in standard HiScribe Reaction)

  1. Thaw the necessary components, keep the T7 RNA Polymerase Mix on ice. 
  2. Mix and pulse-spin in a microfuge to collect the solutions to the bottom of the tubes. 
  3. Set up the reaction at room temperature in the following order:
  4. Component



    Nuclease-free Water

    X µl


    10X Reaction Buffer

    2 µl

    0.5X final

    100 mM ATP

    2 µl

    5 mM final

    100 mM GTP

    2 µl

    5 mM final

    100 mM UTP

    2 µl

    5 mM final

    100 mM CTP

    2 µl

    5 mM final

    100 mM CleanCap® AG or AG (3’ OMe) (N-7113 or N-7413)

    1.6 µl

    4 mM final

    Linear Template DNA

    X µl

    1 µg total

    T7 RNA Polymerase Mix

    4 µl



    40 µl


  5. Mix thoroughly by pipet and pulse-spin in a microfuge.  Incubate at 37°C for 2 hours in a dry air incubator or PCR machine to prevent evaporation.
  6. Follow the protocol provided with the HiScribe T7 High Yield RNA Synthesis Kit (NEB #E2040S) for DNase treatment and RNA purification.