Please find the following protocol:

Transient Transfection and Spin Infection Protocol


Protocol:

1. Plate packaging cells at 2.5 X 106 cells per 60 mm plate. Or remove a packaging cell line plate (which should be confluent) and split into 3 plates.

2. Next day, combine in one tube 10 μg of DNA and 134 μl 2 M CaCl2 add ddH2O to bring the total volume to 1 ml.

3. To the DNA solution add 1 ml of 2X HBS with oxygenation. Create bubbles in the DNA containing solution with another pipette as the HBS solution is being added.

4. Discard the media from the 60 mm plate containing cells and add 8 ml of fresh Cell Line Media.

5. Slowly add the 2 ml of DNA Solution by dripping some solution into the 60 mm plate and shaking the plate to mix the DNA into the media.

6. Incubate for 8 hrs at 37°C.

7. Discard the media from the 60 mm plate containing cells; add 5 ml of fresh Cell Line Media and continue incubation overnight.

8. Put cell line to be infected at a concentration for optimal growth.

9. Forty-eight hours after transfection harvest the virus supernatant from packaging cell line and centrifuge down to remove cells (1,200 X g for 5 min.). Save the virus supernatant for Step #11. Freeze any unused virus supernatant at -80°C. Efficiency of the frozen stock of virus for infection will be about half of what it was for the fresh supernatant.

10. Harvest 5 to 10 x 105 cells to be infected by centrifugation (1,500 X g) and decant the supernatant.

11. Add 2 μl of Polybrene to 2 ml of virus supernatant.

12. Resuspend cells to be infected with the 2 ml of virus supernatant containing Polybrene.

13. Transfer cells and virus supernatant to a 6 well plate.

14. Centrifuge at 1,200 X g for 2 hours at room temperature.

15. Remove most of the viral supernatant from the well and add 4 ml of media to cells.

16. Two days later, the cells are ready to be analyzed or put cells into selection if you want to obtain stable clones.



Solutions:

2 M CaCl2.........Solution is stable at -20°C for approx. 6 months
HBS Buffer (2X),,,,,,,,,,,,,,,,,,,,,,,,,,,,Adjust the pH to 7.05 to 7.15
...............Solution is stable at -20°C for approx. 6 months
........Accurate pH and the amount of phosphate are crucial
................................................................42 mM HEPES
.................................................................Filter sterilize
.....................................................................10 mM KCl
..................1.8 mM Sodium Phosphate Dibasic (Na2HPO4)
...................................................................0.27 M NaCl
Polybrene............4 mg/ml Hexadimethrine Bromide (Polybrene)
Cell Line Media


Bioreagents and Chemicals:

HEPES
Hexadimethrine Bromide
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Calcium Chloride


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1461