In vitro transcription with T7 RNA polymerase: Difference between revisions
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Adapted from: Cazenave, C., Uhlenbeck, O.C. ''Proc. Natl. Acad. Sci. USA'' '''1994''', ''91'', 6972–6976. | Adapted from: Cazenave, C., Uhlenbeck, O.C. ''Proc. Natl. Acad. Sci. USA'' '''1994''', ''91'', 6972–6976. | ||
T7 RNAP=T7 RNA polymerase | |||
==Protocol== | ==Protocol== | ||
In progress... | In progress... | ||
===Template DNA=== | ===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 AT'''T AAT ACG ACT CTA TA'''-3´ (promoter sequence in bold). This primer is also useful for sequencing plasmid that have the T7 RNAP promoter. | |||
===Transcription buffer=== | ===Transcription buffer=== | ||
1X buffer: | 1X buffer: | ||
Revision as of 09:24, 17 June 2005
Adapted from: Cazenave, C., Uhlenbeck, O.C. Proc. Natl. Acad. Sci. USA 1994, 91, 6972–6976.
T7 RNAP=T7 RNA polymerase
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 CTA TA-3´ (promoter sequence in bold). This primer is also useful for sequencing plasmid that have the T7 RNAP promoter.
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.
