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The Laboratory of Donald Rio at the University of California, Berkeley
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| This protocol describes the incorporation of a radioactive nucleotide into RNA using T7 RNA polymerase. The gene of interest is cloned into an appropriate vector containing a T7 promoter directly upstream of the gene (several commercially supplied plasmids are available for this purpose). Cleavage of the plasmid with a restriction enzyme downstream or 3' to the gene ensures that the transcripts terminate appropriately. Inclusion of GpppG, or "cap", allows the transcript to be efficiently translated in an in vitro translation system such as rabbit reticulocyte lysate (see protocol on In Vitro Transcription and Translation Using the Coupled Reticulocyte System). The transcripts are gel purified to remove any truncated transcipts. |
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RNA is very sensitive to nuclease activity. Precautionary techniques, such as including diethyl pyrocarbonate (DEPC) treatment of solutions, can limit RNA degradation. Refer to Working with RNA in the BioTools section of the website
1. Cleave the plasmid DNA at a restriction site at the 3' end of the gene of interest (see protocol on Restriction Enzyme Digest of DNA).
2. Add the following reagents in order at room temperature:
For 0.5X reaction
12.125 μl of ddH2O
0.5 μl of 0.5 M DTT
1 μl of BSA Solution
2.5 μl of 10X rNTPs (see Hint #1)
1.25 μl of GpppG (see Hint #1)
0.5 μg/μl of DNA (prepared in Step #1)
40 μCi/μl of α-[32P]-rUTP (CAUTION! see Hint #2)
2.5 μl of 10X T7 Buffer
(X) Units of T7 RNA Polymerase (see Hint #4)
Bring the final volume to 25 μl using ddH2O.
3. Incubate the reaction mixture at 37°C for 1 to 4 hours (see Hint #3).
4. Quench the reaction with an equal volume of 5 M Ammonium Acetate.
5. Allow the reaction to cool to room temperature and add an equal volume of Phenol (CAUTION! see Hint #2), mix well, centrifuge to separate the phases, and transfer the aqueous phase (upper layer) to a new microcentrifuge tube, avoiding contamination with the organic phase.
6. To the aqueous phase, add 2.5 volumes of -20°C 100% Ethanol and chill the reaction in a Dry Ice/Ethanol bath (see protocol on Preparation and Use of a Dry Ice / Ethanol Bath) for 10 min.
7. Microcentrifuge the solution for 10 min at 4°C to pellet the RNA.
8. Decant the supernatant and add 500 μl of -20°C 70% Ethanol and mix by inversion.
9. Microcentrifuge the solution for 5 min at 4°C to pellet the RNA.
10. Decant the supernatant, dry the pellet by inverting the tubes over a paper towel and allow to air dry for a couple of minutes.
11. Resuspend the RNA pellet in 25 to 50 μl of Sample Buffer and mix well.
12. Place the tubes in a boiling water bath for no longer than 90 sec.
13. Pre-run a denaturing Polyacrylamide gel (16 X 0.15 cm using 0.5 cm well or larger) at 350 V for 15 min (see protocol on Polyacrylamide Gel Electrophoresis), load samples and electrophoresis as needed for RNA separation (determine length of time empirically).
14. Identify the transcript by autoradiography (see protocol on Autoradiography) and excise the Polyacrylamide gel slice containing the band of interest from the gel. Transfer the gel slice containing labeled RNA to microcentrifuge tubes and add 500 to 800 μl Elution Buffer.
15. Incubate the gel sections in elution buffer overnight at 4°C shaking at approximately 200 RPM.
16. Microcentrifuge the tubes to separate the phases for 10 sec and transfer the supernatant to a fresh microcentrifuge tube (see Hint #5).
17. To decrease the total volume of the supernatant, add an equal (or 2X) volume of Butanol, mix well, microcentrifuge to separate the phases and save the aqueous phase (upper layer).
18. To the aqueous phase, add an equal volume of Saturated Phenol, mix well, microcentrifuge to separate the phases and save the aqueous phase (upper layer).
19. To the aqueous phase, add an equal volume of SEVAG, mix well, microcentrifuge to separate phases and save the aqueous phase (upper layer).
20. Repeat Step #18. To the supernatant, add 2.5 volumes of -20°C 100% Ethanol and chill the reaction in a Dry Ice/Ethanol bath for 10 min.
21. Microcentrifuge the tubes at maximum speed for 10 min at 4°C to pellet the RNA.
22. Decant the supernatant and add 500 μl of -20°C 70% Ethanol and mix by inversion.
23. Microcentrifuge the tubes to pellet the RNA for 5 min at 4°C.
24. Decant the supernatant, dry the pellet by inverting the tubes over a paper towel and allow to air dry for a couple of minutes.
25. Resuspend the RNA pellet in 50 to 200 μl of ddH2O (or buffer of your choice) and mix by vortexing to ensure resuspension.
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| T7 Buffer (10X) |
| 0.5 M NaCl
Store at -20°C
0.02 M Spermidine
0.06 M MgCl2
0.4 M Tris-HCl, pH 7.5
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| 10 mM GpppG |
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| Saturated Phenol |
| Add approximately 1:10 the volume of ddH2O
Carefully remove the upper (aqueous) phase
100% Phenol
Mix well and incubate at room temperature for 12 hr
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| Sample Buffer |
| 10 mM EDTA, pH 8.0
95% Formamide
0.09% Xylene Cyanol FF
0.09% Bromophenol Blue
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| α-[32P]-rUTP |
| 800 Ci/mmole α-[32P]-rUTP
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| SEVAG |
| 24:1 Chloroform:Isoamyl Alcohol
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| rNTPs (10X) |
| 0.5 mM rUTP
5 mM rCTP
5 mM rATP
*For uncapped transcripts use 5 mM rGTP
0.5 mM rGTP*
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| 70% (v/v) Ethanol |
| Store and use at -20°C
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| BSA Solution |
| 2.5 mg/ml Bovine Serum Albumin, RNase free
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| 100% (v/v) Ethanol |
| Store and use at -20°C
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| 0.5 M DTT |
| Prepare DTT just before use
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| 5 M Ammonium Acetate |
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| Elution Buffer |
| 0.1% (w/v) SDS
1 mM EDTA
10 mM Magnesium Acetate
0.5 M Ammonium Acetate
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Spermidine
Magnesium Chloride
Isoamyl Alcohol
GTP
Chloroform
Phenol
Xylene Cyanol FF
Tris-HCl
Bromophenol Blue
SDS
Sodium Chloride
Butanol
Ethanol
α-[32P]-rUTP
Formamide
Urea
Ammonium Acetate
Bovine Serum Albumin, RNase Free
rUTP
DTT
Magnesium Acetate
EDTA
Dry Ice
rCTP
rATP
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1. If preparing uncapped RNA transcripts prepare 10X rNTP solution using 5 mM rGTP instead of 0.5 mM rGTP AND substitute ddH2O for GpppG.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. Although the yield does not generally increase after four hours of incubation, this should be confirmed empirically.
4. The optimal number of units of T7 RNA polymerase to use should be determined empirically to maximize transcript yield. For commercially available enzymes, start by following the manufacturer's instructions.
5. Make sure that you do not transfer any of the Polyacrylamide gel into the supernatant. If a small amount of Polyacrylamide is present, repeat the microcentrifuge step as many times as needed.
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1. Rio, DC. RNA Processing. Curr Opin Cell Biol. 1992;4:444-452.
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