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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 09:06, 21 June 2005
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Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate: | Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate: | ||
P<sub>2</sub>O <sub>7 <sup>-4 + H2O -> 2HPO<sub>4 <sup>-2. Inorganic pyrophosphate is released when a nucleoside triphosphate is incorporated/polymerized into the growing chain. This helps to prevent 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. | P<sub>2</sub>O<sub>7</sub><sup>-4</sup> + H2O -> 2HPO<sub>4</sub><sup>-2</sup>. Inorganic pyrophosphate is released when a nucleoside triphosphate is incorporated/polymerized into the growing chain. This helps to prevent 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 | Make a 0.1 U/µL stock solution in H<sub>2</sub>O and store at -20 ˚C. | ||
===T7 RNA polymerase=== | ===T7 RNA polymerase=== | ||
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===Storing RNA=== | ===Storing RNA=== | ||
*Always store RNA at a neutral pH with some amount of EDTA. I recommend TE buffer (10 mM Tris- | *Always store RNA at a neutral pH with some amount of EDTA. I recommend TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Thinking "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. | Store RNA at -20 ˚C. | ||
