Editing Protein Production using Baculovirus (section)

=== <br>Amplify P1 viral stock in a 6-well plate.<br>  ===

<br>1. On the day of infection, prepare your Sf9 or Sf21 cell suspension and plate cells at 2 X 10<sup>6</sup> cells/well. Incubate cells at room temperature for 1 hour to allow attachment. 

2. After 1 hour, inspect cells under an inverted microscope to verify attachment. 

3. Add the appropriate amount of P1 viral stock to each well. 

4. Incubate the cells for 48 hours in a 27°C humidified incubator. 

5. 48 hours post-infection, collect 2 ml of medium containing virus from each well and transfer to sterile 15 ml snap-cap tubes. Centrifuge the tubes at 500 X g for 5 minutes to remove cells and large debris and to obtain clarified baculoviral stock. 

Note: It is possible to harvest virus at later times after infection (e.g., 72 hours). Optimal harvest times can vary and should be determined for each baculoviral construct. Remember that culture viability will decrease over time as cells lyse. 

6. Transfer the supernatant to fresh 15 ml snap-cap tubes. This is the '''P2''' viral stock. Store at 4 °C, protected from light. For long-term storage, store an aliquot of the P2 stock at –80 °C, protected from light. 

7. Proceed to the next section to determine the titer of P2 viral stock. 

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