SP6 In Vitro Transcription Reaction Protocol (M0207)
- 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
0.5 mM each
RNase Inhibitor (optional)
1 U/µl final
Fresh DTT (optional)
5 mM final
SP6 RNA Polymerase
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- 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).