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.

Contents

Protocol

In progress...

Template DNA

Transcription buffer

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.

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.

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