(source: Jasper Rine Lab.)
A. 1. Digest the gene-containing plasmid DNA to completion with a restriction endonuclease to linearize the template DNA 3' to the gene for both the SP6 and T7 transcription reactions (see Hint #1).
2. "Gel purify" the DNA fragment by running the digest on an Agarose gel (see Protocol on Running DNA on Agarose Gels). Cut the DNA out of the gel, remove the Agarose from the DNA, and clean up the DNA by Phenol extractions (see Protocol on Isolation of DNA from Agarose Gels).
3. Add 2 μl of linearized DNA (approximately 1 μg) to a sterile microcentrifuge tube.
4. Add 66 μl RNAse-free ddH2O
10 μl of 10X T7 Buffer or 10X Transcription Buffer supplied with Polymerase Enzyme
5 μl of 250 mM DTT
10 μl of 5 mM m7G(5')ppp(5')G
4 μl of rNTP Mix
1 μl of RNasin (20 Units/μl)
2 μl of T7 or SP6 RNA Polymerase
5. Incubate for 30 min at 37°C for T7 RNA Polymerase or at 40°C for SP6 RNA Polymerase.
6. Add 1 μl of 100 mM rGTP.
7. Incubate for 1 hr at 37°C or 40°C.
8. Add 4 μl of 0.5 M EDTA, pH 8.0 (final concentration of EDTA should be 20 mM).
9. Add 2.5 volumes of 100% Ethanol and 0.1 volumes of 7.5 M Ammonium Acetate to the RNA.
10. Precipitate the RNA by incubating it on Dry Ice for 30 min.
11. Centrifuge for 10 min at full speed in a microcentrifuge at 4°C.
12. Remove all traces of Ethanol (as it may inhibit an in vitro translation assay) from the sample by evaporation in a Speed-Vac concentrator.
13. Resuspend pellet in 25 μl of RNase-free ddH2O. Use mRNA in an in vitro translation reaction.