Biology Reference
In-Depth Information
13.1.1
Purification from Sf9 cells
13.1.1.1
Amplifying virus
13.1.1.1.1
Buffers, reagents, and equipment
13.1.1.1.1.1
Reagents
Cellfectin transfection kit (Invitrogen, 10362-100, Carlsbad, CA)
6-well plate (Thermo Fisher Scientific, 353224, Agawam, MA)
Petri Dish, 150
15 mm Polystyrene (Thermo Fisher Scientific, 08-757-148,
Agawam, MA)
Sf9 insect cell cultures
Serum-free media—SFM-900 II media (Gibco, 10902, Carlsbad, CA)
HI fetal bovine serum (FBS) (Gibco, 16140, Carlsbad, CA)
Bacmid
13.1.1.1.1.2
Equipment
Shaking incubator
Tabletop centrifuge for 15 ml conicals
13.1.1.1.2
Detailed procedures
Recombinant baculovirus is prepared according to Bac-to-Bac system protocol
(Invitrogen, Carlsbad, CA). All information included here is also found in the
manual.
13.1.1.1.2.1
Transfecting Sf9 cells
1.
First, following the kit protocol, transfect 1
10
6
Sf9 cells in a 6-well plate with
2
m
g of the bacmid DNA using Cellfectin or equivalent Sf9 transfection
reagent. There should be no FBS or antibiotics in the media during the
transfection.
2.
Once cells have been transfected, let the culture incubate for 3-5 h at 27
C.
3.
Change the transfection media on the cells to fresh SFM media with 5% FBS.
4.
Incubate the cells for 72 h or until you see the cells lysing and dying. If you are
not sure if the cells are infected, wait for 5-7 days and then harvest the virus.
Wrap the 6-well plate in a damp towel and cover with a plastic wrap to keep moist
and prevent drying out (this is a humid chamber).
13.1.1.1.2.2
Harvesting P1 Recombinant Baculovirus stock
1.
Transfer media from transfected cells to a 15-ml conical with a sterile 10-ml
pipette.
2.
Centrifuge gently at 1500
g
in a clinical centrifuge for 5 min at room
temperature to remove any remaining cells from the media.
3.
Transfer the supernatant to a new 15-ml conical with a sterile 10-ml pipette.
4.
Wrap the conical in tin foil to protect from the light and store at 4
C. This is the
P1, which is also referred to as the first titer of virus.
13.1.1.1.2.3
Making P2 and P3 Recombinant Baculovirus stocks
1.
Plate 3
10
6
cells in 25 ml in a 150
15-mm Petri Dish.
