In vitro transcription with T7 RNA polymerase: Difference between revisions
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==Protocol== | ==Protocol== | ||
In progress... | |||
===Template DNA=== | ===Template DNA=== | ||
===Transcription buffer=== | ===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=== | ===T7 RNA polymerase=== | ||
Clones of T7 RNA polymerase with an N-terminal His-6 tag are available. (see [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9116496&query_hl=1 He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. ''Protein Expr Purif.'' '''1997''', ''9'', 142–151.]) | Clones of T7 RNA polymerase with an N-terminal His-6 tag are available. (see [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9116496&query_hl=1 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. | 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. | ||
Revision as of 08:33, 17 June 2005
Adapted from: Cazenave, C., Uhlenbeck, O.C. Proc. Natl. Acad. Sci. USA 1994, 91, 6972–6976.
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.
