(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.