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The Laboratory of Donald Rio at the University of California, Berkeley
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1. Seed 2 million Sf9 cells into a 25 cm2 T-flask and allow cells to attach for at least 1 hr.
2. Mix 1 μg of the insect baculovirus vector AcMNPV linear viral DNA and 2 μg of recombinant plasmid DNA in a snap cap polypropylene tube.
3. Prepare 5 ml of HEBS/CT by combining 0.5 ml of 10X HEBS, 75 μl of sonicated calf thymus DNA, 4.425 ml of ddH2O and adjust the pH to 7.05 to 7.1 with 0.1 M NaOH. Filter sterilize and allow the solution to come to room temperature before use.
4. Add 950 μl of HEBS/CT to the DNA mixture and mix by vortexing.
5. Add 50 μl of 2.5 M CaCl2 to the DNA/HEBS/CT mixture drop-wise with constant mixing.
6. Incubate at room temperature for 30 min.
7. Remove the media from the tissue culture T-flask and replace it with 2 ml of Complete Grace's Insect Medium.
8. Add DNA precipitate solution to the cell culture flask drop-wise with mixing and incubate at 37°C for 4 hr.
9. Remove the Grace's media from the flask. and rinse the cells once with Complete Grace's Insect Media.
10. Remove the Grace's Insect Media and replace it with Complete TNM-FH Insect Media.
11. Incubate at 27°C for 3 to 6 days, looking for signs of infection daily (see Hint #1).
12. Harvest the supernatant when the cells are ready and store the transfection supernatant at 4°C.
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| 0.1 M NaOH |
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| CT DNA |
| 1 mg/ml Sonicated Calf Thymus DNA
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| 2.5 M CaCl2 |
| Make 0.5 ml aliquots and store at -20°C
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| 10X HEBS Buffer |
| 200 mM HEPES, Free Acid
7 mM Sodium Phosphate Dibasic (Na2HPO4)
600 mM Glucose
Adjust the pH to 7.1 with fresh 5 M NaOH
Sterile filter and store at -20°C
1.37 M NaCl
50 mM KCl
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| TNM-FH Insect Medium (Complete) |
| TNM-HM Insect Medium
100 U/ml Penicillin
2 mM Glutamine
10% Fetal Calf Serum
100 μg/ml Streptomycin
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| Grace's Insect Medium (Complete) |
| 2 mM Glutamine
10% Fetal Calf Serum
Grace's Insect Medium
100 Units/ml Penicillin
100 μg/ml Streptomycin
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HEPES, Free Acid
TNM-FH Insect Medium
Sodium Phosphate, Dibasic
Potassium Chloride
Grace's Insect Medium
Sodium Chloride
Glutamine
DNA, Calf Thymus
Streptomycin
Penicillin
Calcium Chloride
AcMNPV viral DNA
Fetal Calf Serum
Sodium Hydroxide
Glucose
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1. Some protocols state that the infection takes only 2 days, but with this protocol it actually takes longer.
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