In vitro transcription with T7 RNA polymerase: Difference between revisions

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Adapted from: Cazenave, C., Uhlenbeck, O.C. ''Proc. Natl. Acad. Sci. USA'' '''1994''', ''91'', 6972–6976.
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T7 RNAP=T7 RNA polymerase
'''In vitro T7 transcription''' is the synthesis of RNAs using a T7 promoter and purified enzyme. It is the standard method of making up to several mg of RNAs longer than about 20nts with relatively high quality as compared to solid phase synthesis.


U=units
In vitro transcribed RNAs like those from T7 or similar viral promoters like T3 and SP6 are important components for many molecular biology experiments. They can be used to generate (antisense) RNA probes for blot hybridisation and nuclease protection assays that are more sensitive than randomly primed DNA probes. Modified nucleotides containing isotopes like <sup>32</sup>P or detectable epitopes like DIG can be integrated into the RNA via T7 transcription. Synthesis can be scaled up for microinjection, viral RNA infection studies, in vitro translation, and binding experiments.


==Protocol==
The [[Sauer lab]] has an excellent, detailed protocol: [[Sauer:In vitro transcription with T7 RNA polymerase]].


===Template DNA===
For a detailed description of PCR-based attachment of T7 promoters see [[Making RNA probes with T7 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.
== See also ==
* [[Knight:In vitro transcription|Knight:In vitro transcription with E. coli RNA polymerase]]


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).
== Related OWW pages ==
===Transcription buffer and other components===
* [[Rebuilding T7]]
'''1X buffer:'''
* [[Endy:Dedicated systems/Transcription]]
* vectors with an N-terminal T7 sequence: [http://openwetware.org/images/2/27/PET11a.pdf pET11a-d], [http://openwetware.org/images/0/08/Vector_Map_-_pET-3a.pdf pET-3a-d]
* vector for blue/white screening and T7 transcription [http://openwetware.org/images/1/1c/PETBlue_2.pdf pETBlue]


50 mM Tris-Cl, pH 7.5
== External links ==
* [http://genetics.mgh.harvard.edu/szostakweb/protocols/transcription/index.html T7 transcription protocol by Jonathan Davies of Harvard Uni (1998)]
* [http://www.ambion.com/techlib/basics/transcription/index.html In vitro transcription basics at Ambion]
* [http://en.wikipedia.org/wiki/T7_RNA_polymerase T7 RNA polymerase - Wikipedia], [http://www.fermentas.com/catalog/modifyingenzymes/rnapolymerases.htm RNA polymerases - Fermentas]


15 mM MgCl2 (How do you make superscripts and subscripts?)
[[Category:Protocol]]
 
[[Category:RNA]]
5 mM dithiothreitol (DTT)
[[Category:In vitro]]
 
2 mM spermidine
 
Make 10X stock and store at -20 ˚C.
 
'''10X NTPs'''
 
20 mM each of ATP, CTP, GTP, and UTP
 
Store at -20 ˚C.
 
'''Inorganic pyrophosphatase'''
 
Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate to form orthophosphate:
P2O7-4 + H2O -> 2HPO4-2. 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.
 
Make a 0.1 U/µL stock solution in H2O and store at -20 ˚C.
 
===T7 RNA polymerase===
Clones of T7 RNA polymerase with an N-terminal His-6 tag are available. (see [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9116496&query_hl=1 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
 
??  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
 
?? 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).
 
Add 10U of RNase-free DNase I and incubate at 37 ˚C, 30 min.
 
Add 5 µL of 500 mM EDTA to stop the reaction.
 
Clean-up/process the RNA:
 
*[[phenol/chloroform extraction]] followed by [[nucleic acid precipitation]]
 
*[[acrylamide gel purification of nucleic acids]]
 
*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.

Latest revision as of 08:07, 20 June 2009

back to protocols

In vitro T7 transcription is the synthesis of RNAs using a T7 promoter and purified enzyme. It is the standard method of making up to several mg of RNAs longer than about 20nts with relatively high quality as compared to solid phase synthesis.

In vitro transcribed RNAs like those from T7 or similar viral promoters like T3 and SP6 are important components for many molecular biology experiments. They can be used to generate (antisense) RNA probes for blot hybridisation and nuclease protection assays that are more sensitive than randomly primed DNA probes. Modified nucleotides containing isotopes like 32P or detectable epitopes like DIG can be integrated into the RNA via T7 transcription. Synthesis can be scaled up for microinjection, viral RNA infection studies, in vitro translation, and binding experiments.

The Sauer lab has an excellent, detailed protocol: Sauer:In vitro transcription with T7 RNA polymerase.

For a detailed description of PCR-based attachment of T7 promoters see Making RNA probes with T7 transcription.

See also

Related OWW pages

External links

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