Retrovirus packaging and infection
Reagents
- Packaging cell: LinX and PT67 (clontech) have been tested and both work well. LinX is the default cell line for this protocol.
- 2.5 M CaCl2: 2.5 M CaCl2, 0.01 M HEPES, adjust pH to 5.5.
- 2 X BBS: 50 mM BES, 280 mM NaCl, 1.5 mM NaHPO4, adjust pH to 6.95.
- salmon sperm DNA from Stratagene #201190.
1. Culture packaging cells to about 70-80% confluent, change to fresh medium 1-4 hrs before transfection.
2. Put 15 ug retroviral plasmid DNA with 15 ug salmon sperm DNA into a 1.5 ml eppendorf tube and add H2O to a total volume of 850 ul. We use 15 ug plasmids per 100 mm round plates.
3. Add 150 ul 2.5 M CaCl2 and mix well. Divide the DNA/CaCl2 mixture into two eppendorf tubes, 500 ul each.
4. Bubble in the DNA/CaCl2 mixture by injecting air with a pasture pipette connecting to a pipette-aid (press the discharging button) at slow speed, meanwhile add 500 ul 2 x BBS dropwise to each tube.
5. Incubate the mixture at room temperature for 10 min. After that, you can see very tiny precipitation under microscope.
6. Drop the precipitation mix (2ml in total) into the packaging cell dish, mix with gentle shaking.
7. After 37C incubation overnight, change to fresh culture medium and incubate the packaging cells at 32C for 48 hrs.
8. Collect the culture medium (can be yellow color) into a 15 ml tube. Spin at 1000 RPM (on Beckman GS-6R) for 5 min at room temperature to remove debris and floating packaging cells.
9. Dilute virus supernatant (virus supernatant: fresh medium = 3:1), and add polybrene(Sigma) to a final concentration of 8 ug/ml before adding onto target cell plates (Target cells should be 30 - 50% confluent at the moment of infection).
10. Spin the target cell plates at 1800 RPM (on Beckman GS-6R) for one hr at room temperature. (This step can be skipped with slightly loss of titration.)
11. Incubate the cells at 32C overnight.
12. Change to common medium, incubate at 37C for 24-72 hrs before analysis. Drugs can be added after 24 hrs for infection selection.
Contributed by S. Lin.