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The Laboratory of Donald Rio at the University of California, Berkeley
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A. Primary Amplification
1. After performing a Neutral Red Plaque Assay on the primary amplification after transfection (see appropriate protocol), pick two plaques to amplify for each virus.
2. Using a short, cotton-plugged Pasteur pipette, carefully pick several well-separated plaques by pipetting the Agarose plug into 1 ml Complete TMN-FH insect media in a 2 ml round-bottom freezing vial.
3. Let the plaque diffuse into the media overnight at 4°C.
4. Seed one million cells in one well of a 6-well tissue culture plate and allow the cells attach to the plate for 1 hr at 27°C.
5. Remove the supernatant and add 0.5 ml of the isolated plaque to each well for each single plaque to be amplified. Rock for 1 hr at 27°C.
6. Add 1.5 ml Complete TMN-FH medium. Parafilm the 6-well dish and place it in a zip-lock plastic bag in a cell incubator. Incubate at 27°C for 4 days.
7. Remove the supernatant to a 15 ml conical tube and centrifuge in a clinical centrifuge at medium speed for 5 min.
8. Transfer the supernatant to a 5 ml freezer vial and store at 4°C.
9. Save the cell pellets for a Western blot. Take up the pellets in 200 μl baculovirus PBS and add 200 μl 2X SDS Sample Buffer. Sonicate the cells, boil for 1 min and analyze 5 μl on an SDS-minigel.
B. Secondary Amplification
1. Seed cells at 5 million cells per plate in four 10 cm dishes.
2. Let the cells attach at 27°C for 1 hr.
3. Take 1 ml of the primary plaque amplification and add 3 ml Complete TMN-FH media.
4. Aspirate the media from the four 10 cm plates for each virus.
5. Add 1 ml of the diluted virus to each 10 cm plate.
6. Rock for 1 hr at room temperature.
7. Add 9 ml of Complete TMN-FH media and incubate for 4 to 5 days at 27°C. Place the plates in a zip-locking plastic bag in a cell incubator.
8. Remove the supernatant to a 50 ml conical tube.
9. Centrifuge in a clinical centrifuge at medium speed for 5 min.
10. Transfer the supernatant to a new 50 ml conical tube. Store at 4°C wrapped in aluminum foil to protect it from light (see Hint #1).
11. Save the cell pellets from the four 10 cm plates to assay or purify protein in a pilot experiment. Resuspend the cells in Baculovirus PBS and repellet the cells. Freeze the pellet in Liquid Nitrogen and store at -80°C.
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| SDS Sample Buffer (2X) |
| 40% (v/v) Glycerol
0.1% (w/v) Bromophenol Blue
4% (w/v) 2-Mercaptoethanol or 6% (w/v) DTT
8% (w/v) SDS
250 mM Tris
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| TMN-FH Insect Medium |
| 100 Units/ml Penicillin
100 μg/ml Streptomycin
TMN-FH Insect Medium (Complete)
10% (v/v) Fetal Calf Serum
2 mM Glutamate
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| Baculovirus PBS |
| 140 mM NaCl
pH 6.2
10.5 mM Potassium Phosphate Monobasic (K2HPO4)
40 mM KCl
1 mM Sodium Phosphate Dibasic (Na2HPO4)
There is no need to adjust the pH
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TNM-FH Insect Medium
Glutamine
Potassium Phosphate, Monobasic
DTT
Sodium Phosphate, Dibasic
Streptomycin
Penicillin
2-Mercaptoethanol
Fetal Calf Serum
Glycerol
Nitrogen, Liquid
Bromophenol Blue
Tris
Potassium Chloride
Sodium Chloride
SDS
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1. This stock should now be titered by a Plaque Assay (see appropriate protocol).
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