In vitro transcription with T7 RNA polymerase

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Adapted from: Cazenave, C., Uhlenbeck, O.C. Proc. Natl. Acad. Sci. USA 1994, 91, 6972–6976.

T7 RNAP=T7 RNA polymerase

Contents

Protocol

In progress...

Template DNA

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

1X buffer:

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.

10X NTPs

20 mM each of ATP, CTP, GTP, and UTP

Inorganic pyrophosphatase

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.

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.

Transcription reaction

For a 100 µL reaction ("preparative scale")

10 µL 10X transcription buffer 10 µL 10X NTPs

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