In vitro transcription with T7 RNA polymerase
Adapted from: Cazenave, C., Uhlenbeck, O.C. Proc. Natl. Acad. Sci. USA 1994, 91, 6972–6976.
T7 RNAP=T7 RNA polymerase
PCR product or linearized plasmid (run-off transcription)
If you use a PCR product, make sure there are at least 5 base pairs upstream of the T7 RNAP promoter. The polymerase needs something to bind to. It is a good idea to have a generic T7 promoter primer that you can use to PCR any template that has the promoter. The one I use has the sequence 5´-GAA ATT AAT ACG ACT CAC TAT A-3´ (promoter sequence in bold). This primer is also useful for sequencing plasmids that have the T7 RNAP promoter.
Transcription buffer and other components
50 mM Tris-Cl, pH 7.5
15 mM MgCl2 (How do you make superscripts and subscripts?)
5 mM dithiothreitol (DTT)
2 mM spermidine
Make 10X stock and store at -20 ˚C.
20 mM each of ATP, CTP, GTP, and UTP
Store at -20 ˚C.
Inorganic pyrophosphatase cleave pyrophosphate (PO4-PO4), which is released when a nucleoside triphosphate is incorporated/polymerized, into phosphate (PO4). This helps to fight against any inhibitory effect of having pyrophosphate around (i.e. prevents the "reverse" reaction.) This is an optional component of the transcription reaction. If you leave it out, often you will see something precipitate (white) in your transcription reaction. This is the pyrophosphate.
Make a 0.1 U/µL stock solution in H2O and store at -20 ˚C.
T7 RNA polymerase
Clones of T7 RNA polymerase with an N-terminal His-6 tag are available. (see He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. Protein Expr Purif. 1997, 9, 142–151.)
It is highly recommended that you obtain this clone and purify your own polymerase. The prep is easy, you should obtain a large amount of polymerase with high activity from a single prep, and you will save a lot of money by not buying the polymerase.
For a 100 µL reaction ("preparative scale"):
10 µL 10X transcription buffer
10 µL 10X NTPs
?? µL DNA template (5–10 pmol) *see below for better description
5 µL inorganic pyrophosphatase (0.1 U/µL): 0.005 U/µL final concentration
?? µL T7 RNAP (25 U/µL final concentration)
OK, so this does sound like a ridiculous amount of enzyme to use. You can get away with a lot less if you are still a slave to corporate America and are purchasing your polymerase. However, if you have produced your own enzyme (see note above), this is not a big deal and you will obtain a truckload of RNA.
Incubate reaction at 37 ˚C for 2 hr. (You can get away with less time here, you'll just get less RNA).
Add 10U of RNase-free DNase I and incubate at 37 ˚C, 30 min.
Add 5 µL of 500 mM EDTA to stop the reaction.
Clean-up/process the RNA:
- Qiagen RNeasy mini kit