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  • SP6 In Vitro Transcription Reaction Protocol (M0207)

    Introduction

    RNase-Free Water: X µl
    10X RNAPol Buffer(NEB #B9012S): 8 µl
    Ribonucleotide Solution Mix (NEB #N0466): 0.5 µl (final concentration of 0.5 mM each)
    Template(s) (1-4 µg): Y µl
    Murine RNase Inhibitor (NEB #M0314): 2 µl
    SP6 RNA Polymerase (20 units/µl): 2-4 µl
    Total reaction volume: 80 µl

    Incubate at 40°C for 2-4 hrs.

    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).