Editing Protein Production using Baculovirus (section)

== <br>Preparing the P1 Viral Stock  ==

1. Once the transfected cells from above Step 6, demonstrate signs of late stage infection (e.g., 72 hours post-transfection), collect the medium containing virus from each well (~2 ml) and transfer to sterile 15 ml snap-cap tubes. Centrifuge the tubes at 500 X&nbsp; g for 5 minutes to remove cells and large debris. 

2. Transfer the clarified supernatant to fresh 15 ml snap-cap tubes. This is the P1 viral stock. Store at 4 °C.<br> <br>Optional: filter viral supernatant through a 0.2 μm, low protein binding filter after the low-speed centrifugation step, if desired. 

<br>3. Store viral stocks as follows:<br>Store viral stock at 4 °C, protected from light.<br> If medium is serum-free (e.g., Sf-900 II SFM, Sf-900TM III SFM), add fetal bovine serum to a final concentration of 2%. Serum proteins act as substrates for proteases.<br>For long-term storage, store an aliquot of the viral stock at –80 °C for later reamplification.<br>Do not store routinely used viral stocks at temperatures below 4 °C. Repeated freeze/thaw cycles can result in a 10- to 100-fold decrease in virus titer.