Cellfectin II Protocol for Sf-21 Cells (6-well plate)
A. Plate 8 × 105 log phase cells (grown without antibiotics) per well in antibiotic
free sf-900 media and allow cells to attach for 15min at room temp.
B. Set up the complexes as shown
DNA Mixture
Reagent
Amount
Graces Insect Media 100µl
Plus Reagent
5µl
1 µg Plasmid DNA
????
Vortex this mixture briefly and incubate at room temperature for 15-30 minutes
Cellfectin Mixture
Reagent
Amount
Graces Insect Media 100µl
Cellfectin II
8 µl
Vortex this mixture briefly and incubate at room temperature for 15-30 minutes
C. Combine the diluted DNA with the diluted Cellfectin II (total volume is ~210 μl).
Mix gently and incubate for 5–15 minutes.
D. Add ~210 μl DNA-lipid mixture dropwise onto the cells
Notes: For Sf-900 II SFM or Sf-900™ III SFM: Incubate cells at 27°C for 24–48 hours
and test for transgene expression. Addition of antibiotics to the growth medium is
optional. Incubate cells at 27°C for 24–48 hours and test for transgene expression.