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).