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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 09:24, 17 June 2005
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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: | ||
