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In vitro transcription with T7 RNA polymerase: Difference between revisions
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In vitro transcription with T7 RNA polymerase (view source)
Revision as of 08:33, 17 June 2005
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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. | ||
