baculoFECTIN II Transfection Reagent 150 ul (GWB-300101)
|Datasheets/Manuals||Printable datasheet for baculoFECTIN II Transfection Reagent 150 ul (GWB-300101)|
|Application||1.) One hour prior to the transfection, seed 1x106 Sf9 cells in 2 mL of growth medium (0.5x106 cells/mL) or 1.5x106 Sf21 cells in 2 mL of growth medium (0.75x106 cells/mL) into each 35mm cell culture dish required-1 dish per recombinant virus plus one mock-transfection control and/or a positive-control transfection using the lacZ transfer plasmid in flashBACTM kit.|
Note: Check cells have settled and formed a sub-confluent monolayer before proceeding. While cells are settling, prepare the transfection mixes (steps 2-3).
2.) For each transfection, pipette 0.1mL serum-free, antibiotic-free medium into a sterile tube (preferably a disposable 1.5mL tube). Add 100ng of baculovirus DNA (e.g. 5 uL flashBACTM DNA at 20 ng/uL) and either 500ng transfer vector DNA containing the gene of interest or 500ng of control transfer plasmid DNA (as supplied in the flashBACTM kits: 5 uL at 100ng/uL) and mix gently to avoid shearing the DNA. In the mock-transfection control, omit the DNA from the medium.
3.) The baculoFECTIN II reagent should be gently vortexed for 5 seconds before adding 1.2uL to each tube containing the transfection mixture. Mix gently and incubate at room temperature for 15-20 min to allow the nanoparticle-DNA complexes to form.
Note: During this incubation stage the solution may appear cloudy due to the baculoFECTIN II interaction with the media. This does not affect the transfection efficiency of baculoFECTIN II.
4.) Remove 1mL of culture medium from the 35mm dishes of cells using a sterile pipette, ensuring that the cell monolayer is not disturbed (leaving 1mL in the dish).
5.) Add the 0.1mL baculoFECTIN II/DNA transfection mixture drop-wise into the center of a dish of cells; repeat for additional viruses and the control samples as needed.
6.) Incubate the dishes in a sandwich box overnight at 28°C.
7.) After this time, add 1mL of your preferred insect cell culture growth medium to each dish (there is no need to remove the transfection reagent) so that each dish has 2mL medium and continue to incubate at 28°C. During this time recombinant virus particles will form and be released into the culture medium by budding.
Note: The 1mL of insect cell culture growth medium added at this stage could be supplemented with antibiotic if preferred e.g. 200 units/mL of penicillin and 200 ug/mL of streptomycin. At this stage a fine precipitate may form on the cells. This is normal and does not affect the cells or transfection efficiency.
8.) At 5 days post-transfection, harvest the 2mL culture medium (containing recombinant virus) into a sterile tube and store in the dark at 4°C (e.g. wrap in foil).
Note: This is your seed stock (P0) of recombinant virus, which can be used to amplify a P1 virus stock by infecting a 50mL Sf9 culture (2x106 cell/mL) with 0.5mL of the seed stock. The P1 virus stock should be harvested 4-5 days post infection and the titre of the virus determined.
9.) If the pAcRP23.lacZ positive control transfer vector supplied with the flashBACTM kit has been used to make recombinant virus, the infected cells can be stained using X-gal. Add 1mL of appropriate insect cell culture medium (or PBS) containing 15uL X-gal (2% w/v in DMF) and incubate at 28°C. After ~5 hours, the cells and culture medium will appear blue in color, confirming the production of recombinant virus expressing lacZ.
|Reconstitution and Storage||Store tightly capped at -20°C.|
- Reconstitution & Storage Instructions
- Western Blotting/Immunoblotting (WB/IB) Protocol
- Immunohistochemistry (IHC) Protocol
- Immunocytochemistry (ICC) Protocol
- Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol
- Blocking Peptide Competition Protocol (BPCP)
- Immunoprecipitation (IP) Protocol
- Antibody Array (AA) Protocol
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