Please find the following protocol:

Amplification of Baculovirus Plaques and Preparation of High Titer Virus Stocks



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.




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. his
stock should now be titered by a Plaque Assay.

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.



Reference Links:

http://www.bio.com/protocolstools/protocol.jhtml?id=p608