| http://axon.med.harvard.edu/~cepko/protocol/mike/B1.html
This is a modified version of the pharmingen Baculogold kit according to Brian Dynlacht.
Solutions
Reagents Needed
HINKS insect medium (Gibco)
Fetal calf serum (HyClone)
Penn/Strep (Gibco)
L-Glutamine (Gibco)
Baculogold transfection kit (Pharmingen)
SF-9 cells and Hi-5 cells
27-28° incubator
Procedure
• Dislodge rapidly growing Sf9 cells by scraping or a sharp blow to the flask. Count the cells and plate 2x106 cells in a 6 cm dish (4-6 ml).
• Allow the cells to adhere for 5 minutes at room temperature.
• Prepare the DNA for transfection by mixing 2 ml baculogold DNA with 2 mg shuttle vector containing the cDNA of interest. Allow to stand for 5 minutes at room temperature.
• Add 1 ml Buffer B to the DNA mixture.
• Aspirate the medium from the cells and add 1 ml Buffer A to the plate.
• Add the buffer B DNA mixture dropwise to the plate containing Sf-9 cells in Buffer A.
• Incubate for 4 hours at 27-28°, wash with medium once and add 3 ml medium. Incubate for 5 days at 27°.
• Harvest the supernatant containing the baculovirus and spin out the cells. Store at 4° for short term storage or add DMSO to 10% and store at -80°.
• To amplify the virus split the Sf-9 cells the night before to about 50-60% confluent and in the morning add 0.5-1.0 ml of viral stock from the transfection.
• 3 days later, harvest the supernatant and repeat with fresh Sf-9 cells.
• Monitor the transfected Sf9 cells for protein production and repeat until significant amounts of protein can be detected (up to 5 amplifications).
• When a high titer viral stock is aquired following amplification, infect Hi-5 cells with 1-2 ml of supernatant per 10 cm dish of Hi-5 cells and harvest protein after 2-3 days.
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