| Contributor: |
The Laboratory of J. Michael Bishop at the University of California, San Francisco
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| This protocol describes the production of a dot blot membrane spotted with RNA isolated from virus particles. |
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1. Prepare an overnight viral infection of a confluent 9 cm dish of cells. Collect approximately 9 ml of media supernatant containing the virus particles.
2. Centrifuge the media at 4°C for 10 minutes at approximately 2,000 X g in a table-top centrifuge to collect supernatant containing the virus particles (half-life of a virus is approximately 4 hr, see Hint #1).
3. Centrifuge the supernatant at 4°C for 2 hours at 169,000 X g (37,000 rpm using an SW40 or SW41 rotor).
4. Carefully decant the supernatant.
5. Add 400 μl of STE/0.2% SDS and 5 μl of Yeast tRNA to the virus pellet.
6. Add an equal volume of Phenol:Chloroform, mix well, microcentrifuge for 3 min at full speed to separate the phases and save the aqueous phase (upper phase).
7. Repeat the Phenol:Chloroform extraction of the aqueous phase (Step #6) 2 more times (3 times total).
8. To the aqueous phase, add an equal volume of Chloroform, mix well, microcentrifuge for 3 min at full speed to separate the phases and save the aqueous phase (upper phase).
9. To the aqueous phase add 0.1 volume of 3 M Sodium Acetate and an equal volume of 100% Ethanol, mix well, and microcentrifuge for 10 min at full speed to pellet the RNA.
10. Discard the supernatant and dry the RNA by microcentrifugation under vacuum or air dry.
11. Resuspend the RNA in 20 μl of TE Buffer.
12. Cut a Nitrocellulose Membrane to the size of the dot blot apparatus. Presoak the membrane in 2X SSC solution for approximately 2 min.
13. Soak the Nitrocellulose Membrane in 20 X SSC for 15 min, blot dry on a piece of Whatman filter paper or paper towel, and dry under a light lamp.
14. Incubate 5 μl of the prepared viral RNA at 65°C for 5 min.
15. Snap cool the solution by placing it in an ice bath (0 to 4°C) for 5 min.
16. Assemble the dot blot apparatus and Nitrocellulose Membrane following the manufacturer's instructions for preparing apparatus.
17. Load the RNA samples immediately onto Nitrocellulose Membrane in the dot blot apparatus under vacuum.
18. After all liquid has been pulled through the membrane, bake the Nitrocellulose Membrane at 80°C for 2 hr under vacuum.
19. Follow directions in the protocol on RNA Northern Blots beginning at the prehybridization step.
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| SSC (2X) |
| 9 parts dd H2O
1 part SSC (20X)
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| SSC (20X) |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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| TE |
| 10 mM Tris-HCl, pH 8.0
1 mM EDTA
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| 3 M Sodium Acetate, pH 5.2 |
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| Phenol:Chloroform |
| 25:24:1 (v/v/v) Phenol:Chloroform:Isoamyl Alcohol (CAUTION See Hint #2)
Store at 4°C in dark glass bottle.
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| Yeast tRNA |
| Extract solution 3 times using Phenol. (CAUTION See Hint #2)
10 mg/ml Yeast tRNA
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| STE/0.2% SDS |
| Prepared in STE buffer.
0.2% (w/v) SDS
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| STE Buffer |
| 1 mM EDTA, pH 8.0
10 mM NaCl
10 mM Tris, pH 7.5
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SDS
tRNA, Yeast
EDTA
Sodium Citrate
Tris
Sodium Chloride
Isoamyl Alcohol
Chloroform
Phenol
Ethanol
Sodium Acetate
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1. At this stage it is acceptable to freeze the supernatant at -70°C. Virus viability decreases 3 to 5 times with each freeze thaw.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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