Baculovirus Transfection Using Calcium C

Description
Baculovirus Transfection Using Calcium Chloride

Procedure
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 ìg of the insect baculovirus vector AcMNPV linear viral DNA and 2 ìg 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 ìl 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 ìl of HEBS/CT to the DNA mixture and mix by vortexing.

5. Add 50 ìl 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.

Recipes
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 ìg/ml Streptomycin

Grace's Insect Medium (Complete) 2 mM Glutamine
10% Fetal Calf Serum
Grace's Insect Medium
100 Units/ml Penicillin
100 ìg/ml Streptomycin

Supplies

Tips
1. Some protocols state that the infection takes only 2 days, but with this protocol it actually takes longer.
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Baculovirus Transfection Using Calcium C