Home Protocols SP6 In Vitro Transcription Reaction Protocol (M0207)

SP6 In Vitro Transcription Reaction Protocol (M0207)

  1. Set up the following reaction on ice
    (Please note that enzyme should always be added last):

    COMPONENTS 20 µl REACTION FINAL CONC. OR AMOUNT
    RNAPol Reaction Buffer

    2 µl

    1X

    NTP

    X µl

    0.5 mM each

    H2O

    X µl

    		 
    Template DNA

    X µl

    1 µg

    RNase Inhibitor (optional)

    0.5 µl

    1 U/µl final

    Fresh DTT (optional)

    X µl

    5 mM final

    SP6 RNA Polymerase

    2 µl

    		 
  2. Gently mix the reaction by pipetting up and down and microfuge briefly.

  3. Incubate at 37°C for 1 hour. For shorter transcripts, incubate at 37°C for 2-16 hours.

For analysis of transcription run 1 µl of transcription reaction on a 1% agarose gel.

Note: Higher yields of RNA may be obtained by raising rNTP concentrations up to 4 mM each. MgCl2 concentrations should also be increased up to 20mM (4 mM above the total rNTP concentration of 16 mM).