Invitrogen Bac to Bac Expression System
Invitrogen Bac to Bac Expression System
Invitrogen Bac to Bac Expression System
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Performing a Viral Plaque Assay, continued<br />
Plaque<br />
Purification<br />
You may generate a viral s<strong>to</strong>ck from a single viral clone by plaque purifying<br />
your baculovirus, if desired. Use a pro<strong>to</strong>col of your choice or the procedure<br />
below.<br />
Materials Needed<br />
• Plate containing well-spaced viral plaques (from Plaque Assay Procedure,<br />
Step 11, page 36; o not stain plates with Neutral Red)<br />
• Log phase Sf9 or Sf21 cells at greater than 95% viability<br />
• Sterile Pasteur pipette and bulb<br />
Procedure<br />
1. Follow Steps 1-3 in the Plaque Assay Procedure, page 36 <strong>to</strong> seed Sf9 or Sf21<br />
cells.<br />
2. Using a sterile Pasteur pipette and bulb, carefully pick a clear plaque and<br />
transfer the agarose plug (containing virus) <strong>to</strong> a 1.5 ml microcentrifuge tube<br />
containing 500 µl of complete growth medium. Mix well by vortexing.<br />
3. Add 100 µl of the agarose plug solution <strong>to</strong> each well.<br />
4. Incubate the cells in a 27ºC humidified incuba<strong>to</strong>r for 72 hours.<br />
5. Collect the medium containing virus from each well (~2 ml) and transfer <strong>to</strong><br />
sterile 15 ml snap-cap tubes. Centrifuge the tubes at 500 x g for 5 minutes <strong>to</strong><br />
remove cells and large debris.<br />
6. Transfer the clarified supernatant <strong>to</strong> fresh 15 ml snap-cap tubes. This is your<br />
plaque-purified viral s<strong>to</strong>ck.<br />
7. Proceed <strong>to</strong> Amplifying Your <strong>Bac</strong>uloviral S<strong>to</strong>ck, page 31.<br />
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