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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 12:40, 17 June 2005
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20 mM each of ATP, CTP, GTP, and UTP | 20 mM each of ATP, CTP, GTP, and UTP | ||
Store at -20 ˚C. | |||
'''Inorganic pyrophosphatase''' | '''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. | 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. | ||
Make a 0.1 U/µL stock solution in H2O and store at -20 ˚C. | |||
===T7 RNA polymerase=== | ===T7 RNA polymerase=== | ||
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===Transcription reaction=== | ===Transcription reaction=== | ||
For a 100 µL reaction ("preparative scale") | For a 100 µL reaction ("preparative scale"): | ||
10 µL 10X transcription buffer | 10 µL 10X transcription buffer | ||
10 µL 10X NTPs | 10 µL 10X NTPs | ||
?? µL DNA template (5–10 pmol) *see below for better description | |||
5 µL inorganic pyrophosphatase (0.1 U/µL): 0.005 U/µL final concentration | |||
?? µL T7 RNAP (25 U/µL final concentration) | |||
OK, so this does sound like a ridiculous amount of enzyme to use. You can get away with a lot less if you are still a slave to corporate America and are purchasing your polymerase. However, if you have produced your own enzyme (see note above), this is not a big deal and you will obtain a truckload of RNA. | |||
Incubate reaction at 37 ˚C for 2 hr. (You can get away with less time here, you'll just get less RNA). | |||
