Co-transfect flashBAC™ & transfer vector into Sf9 cells
2 ml P1 stock 1x107-5x107 pfu/ml
500 ml P2 stock > 1x108 pfu/ml
Transform DNA into DH10Bac E. coli = transposition into bacmid
Blue-white selection of recombinant E. coli on antibiotic plates
Verify recombinant colonies by PCR
Amplify and purify DNA, transfect into 35 mm2 dish of Sf9 cells
2 ml P1 stock, 2x107 - 4x107 pfu/ml
500 ml P2 stock > 1x108 pfu/ml
Gene of interest cloned into Gateway® entry vector
Obtain seed stock
Complete plaque assay of seed stock prior to amplification
to determine titre (optional)
Obtain high titre virus stock
(day 10 if plaque assay not done)
Co transfect BacPAK and transfer vector into cells
Seed stock obtained (2 ml)
Plaque purification completed - 8 days
Verify recombinant plaques by PCR, infect flask for P1 stock
Obtain P1 stock
Obtain P2 stock (500 ml)
Co-transfect BacVector 3000 & transfer vector into Sf9 cells
2 ml P0 stock, 1x106 - 1x107 pfu/ml
2 rounds plaque purification = 7 days
Verify recombinant plaques by PCR, infect 25 cm2 flask for P1 stock
5 ml P1 stock, 1x106 - 1x107 pfu/ml
500 ml P2 stock > 1x108 pfu/ml
Co-transfect Bac-n-Blue & transfer vector into Sf9 cells
2 ml P1 stock 1x106-1x107 pfu/ml
2 rounds plaque purification = 7 days
Verify recombinant plaques by PCR, infect 25 cm2 flask
for P1 stock
Amplify 5 ml P1 stock 1x106-1x107 pfu/ml
500 ml P2 stock
> 1x108 pfu/ml