Editing Expression of HA NA using Baculovoirus (section)

=== <br> Preparation of working stocks<br> ===

<br> 1. Plate 5 x 105 Sf9 cells/cm2 in a T175 cm2 flask and let sit for 20 min allow them to attach to the dish. Replace complete TNM-FH media with TNM-FH media containing 3% FBS (and 1% Pen-Strep, 0.1% Pluronic F68).<br>

<br> 2. Infect cells with 100 μl of the rescue supernatant. Incubate in a 28 °C incubator without CO2 for 6 days and check occasionally if cells get infected.<br>

<br> 3. Spin at 2,000 x g at room temperature for 5 min and collect supernatant. This is now your P2 stock. This stock can be stored indefinitely at 4 °C. Repeat the infection procedure using 100 μl of the P2 stock to infect cells. The resulting stock is called P3 or working stock, which can be stored at 4 °C or used directly for protein expression. The P3 stock usually contains 107-109 plaque forming units/ml.

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