Knight:In vitro transcription protocol

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Solutions/reagents:

  • repressor
  • PCR template
  • 5X E. coli RNA polymerase transcription buffer
  • TCEP
  • 2.5 mM each NTP
  • RNase-free water
  • E. coli RNA polymerase holoenzyme
  • DNaseI buffer
  • DNase

Equipment:

  • Incubator
  • Reaction tubes

Steps:

  1. Prepare template DNA

    1. Generate linearized template via PCR. Do a 100 ?L reaction using VF2 and VR.
      Can be done once, frozen and reused.

  2. Option 1: Preincubate repressor and DNA

    1. Measure out 20 µl of repressor into reaction tube (1).
      Add 2 µl of PCR template.
      Do the same for relevant controls.

    2. Incubate at room temperature for 2 hrs.

    3. Use the following table as a checklist for preparing the reaction in reaction tube (2):

      <thead></thead><tbody></body>
       repressor-DNA mixture5X E. coli RNA polymerase transcription bufferTCEP2.5 mM each NTPE. coli RNA polymerase holoenzymeRNase-free water
      Reaction22 µl10 µl0.5 µl10 µl2.5 µl5 µl

    4. Incubate at 37°C for 1 hr.

    (or)

    Option 2: Set up transcription reaction

    1. Use the following table as a checklist for preparing the reaction in reaction tube (2):

      <thead></thead><tbody></body>
       RNase-free water5X E. coli RNA polymerase transcription bufferTCEP2.5 mM each NTPPCR templateE. coli RNA polymerase holoenzyme
      Reaction25 µl10 µl0.5 µl10 µl2 µl2.5 µl

    2. Incubate at 37°C for 1 hr.

    3. DNase treatment (Optional)
    This step hasn't been tried.
    An optional step is to treat the reaction with RNase free DNaseI to remove the template DNA.
    Measure out 6 µl of DNaseI buffer into reaction tube (2).
    Add 3 µl of RNase-free water.
    Add 1 µl of DNase.
    Incubate at 37°C for 1 hr.
    Store at 75°C for 10 mins.
    This is to heat-inactivate the enzyme.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 3 hrs, 10 mins

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