In vitro transcription with T7 RNA polymerase: Difference between revisions
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PCR product or linearized plasmid (run-off transcription) | 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 CAC TAT A'''-3´ (promoter sequence in bold). This primer is also useful for sequencing | 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 CAC TAT A'''-3´ (promoter sequence in bold). This primer is also useful for sequencing plasmids that have the T7 RNAP promoter. | ||
===Transcription buffer=== | ===Transcription buffer and other components=== | ||
1X buffer: | '''1X buffer:''' | ||
50 mM Tris-Cl, pH 7.5 | 50 mM Tris-Cl, pH 7.5 | ||
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Make 10X stock and store at -20 ˚C. | 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=== | ===T7 RNA polymerase=== | ||
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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. | ||
===Transcription reaction=== | |||
For a 100 µL reaction ("preparative scale") | |||
10 µL 10X transcription buffer | |||
10 µL 10X NTPs | |||
Revision as of 12:27, 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 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
