Transfection of SF9 cells with Recombinant Bacmid DNA
(from Gibco-BRL Bac-to-Bac Instruction manual)
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Seed 9 x 105 cells per well in 6-well plate in 2ml Grace’s media supplemented with
10% FBS and antibiotic-antimycotic. Use only cells from 24h-old suspension
culture in mid-log phase with a viability of >97%. Rock the plates (do not swirl)
back-forth and from side to side to distribute the cells evenly.
Allow cells to attach at 27C for at least 1 h.
Prepare the following solution in eppendorf or 12 x 75-mm sterile polypropylene
tubes:
Solution A: For each transfection, dilute ~10l of “Quick and Dirty” prep of
Bacmid DNA into 100l SF-900 SFM without antibiotics.
Solution B: For each transfection, dilute 10-15l CellFECTIN Reagent into
100l SF-900 SFM without antibiotics. Note: CellFECTIN Reagent is a lipid
suspension that may settle with time. Mix thoroughly by inverting the tube 510 times before removing a sample for transfection to ensure that a
homogeneous sample is taken.
Combine the two solutions, mix gently, and incubate for 15-45 min at room
temperature.
Wash the cells once with 2 ml of SF-900 SFM without antibiotics.
For each transfection, add 0.8 ml of SF-900 SFM to each tube containing the lipidDNA complexes. Mix gently. Aspirate wash media from cells and overlay the
diluted lipid-DNA complexes onto the cells.
Incubate cells for 5 h in a 27C incubator.
Remove the transfection mixtures and add 2-2.5ml of Grace’s media containing FBS
and antibiotic. Put the plate in closed box with wet kimwipe on the bottom to
prevent media evaporation. Incubate cells in a 27C incubator for 4-5 days.
Harvest virus from cell culture medium, call it P0. Use1 ml for further amplification
of virus in 100ml SF9 cells. Use the cells to run the SDS-page to verify
expression of the recombinant protein.
From the initial transfection, viral titers of 2 x 107 to 4 x 107 pfu/ml can be expected.
For amplifying viral stocks, infect a cell culture at Multiplicity of Infection (MOI)
of 0.01 to 0.1. Harvest virus at 72-96 h post-infection. That will result in
approximately 100-fold amplification of the virus.
Store virus at 4C, protected from light. For long-term storage of virus, the addition
of FBS to a final concentration of at least 2% is recommended. Storage of an
aliquot of the viral stock at -70C is also recommended.