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Baculovirus Transfection Using Calcium Chloride

Baculovirus Transfection Using Calcium Chloride
Contributor: The Laboratory of Donald Rio at the University of California, Berkeley
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.

0.1 M NaOH
CT DNA   1 mg/ml Sonicated Calf Thymus DNA
2.5 M CaCl2   Make 0.5 ml aliquots and store at -20°C
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
TNM-FH Insect Medium (Complete)   TNM-HM Insect Medium
100 U/ml Penicillin
2 mM Glutamine
10% Fetal Calf Serum
100 μg/ml Streptomycin
Grace's Insect Medium (Complete)   2 mM Glutamine
10% Fetal Calf Serum
Grace's Insect Medium
100 Units/ml Penicillin
100 μg/ml Streptomycin
BioReagents and Chemicals
HEPES, Free Acid
TNM-FH Insect Medium
Sodium Phosphate, Dibasic
Potassium Chloride
Grace's Insect Medium
Sodium Chloride
DNA, Calf Thymus
Calcium Chloride
AcMNPV viral DNA
Fetal Calf Serum
Sodium Hydroxide
Protocol Hints
1. Some protocols state that the infection takes only 2 days, but with this protocol it actually takes longer.