In vitro transcription with T7 RNA polymerase

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==Protocol==
==Protocol==
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In progress...
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===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.
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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
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??  µL  DNA template (5–10 pmol) *see below for better description
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??  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
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Determine the concentration of your RNA. [[Quantification of nucleic acids]]
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*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?
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Store RNA at -20 ˚C.

Revision as of 15:01, 17 June 2005

Adapted from: Cazenave, C., Uhlenbeck, O.C. Proc. Natl. Acad. Sci. USA 1994, 91, 6972–6976.

T7 RNAP=T7 RNA polymerase

U=units

Contents

Protocol

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 ATT 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

1X buffer:

50 mM Tris-Cl, pH 7.5

15 mM MgCl2 (How do you make superscripts and subscripts?)

5 mM dithiothreitol (DTT)

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 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

Clones of T7 RNA polymerase with an N-terminal His-6 tag are available. (see 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:

  • 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.

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