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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 13:01, 17 June 2005
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==Protocol== | ==Protocol== | ||
===Template DNA=== | ===Template DNA=== | ||
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 plasmids that have the T7 RNAP promoter. | 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. | ||
I generally recommend using 5–10 pmol of DNA template in a 100 µL transcription reaction. Does this mean you need to determine the concentration of your DNA? Not really, a reasonable estimate is good enough. For a 5000 base pair plasmid, 5 pmol is approximately 16 µg of DNA. For a PCR reaction, estimate the total number of pmols in your PCR by assuming that the reaction went to completion and half of your primers were used up (ex. a reaction with 50 pmol of each primer should yield approximately 25 pmol of extended product). | |||
===Transcription buffer and other components=== | ===Transcription buffer and other components=== | ||
'''1X buffer:''' | '''1X buffer:''' | ||
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10 µL 10X NTPs | 10 µL 10X NTPs | ||
?? µL DNA template (5–10 pmol) *see | ?? µL DNA template (5–10 pmol) *see above for better description | ||
5 µL inorganic pyrophosphatase (0.1 U/µL): 0.005 U/µL final concentration | 5 µL inorganic pyrophosphatase (0.1 U/µL): 0.005 U/µL final concentration | ||
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*Qiagen RNeasy mini kit | *Qiagen RNeasy mini kit | ||
Determine the concentration of your RNA. [[Quantification of nucleic acids]] | |||
*Always store RNA at a neutral pH with some amount of EDTA. I recommend TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA). Thinking that "I'll just put it in water" is a bad idea for RNA (and DNA and proteins and...). Do you really know what's in that water? | |||
Store RNA at -20 ˚C. | |||
