Polybrene Infection / Transfection Reagent Protocol

Plate cells at approximately 50% confluence in complete growth medium.
After 18 to 24 hours post plating, prepare the DNA-Medium-Polybrene solution and use immediately (see Protocol 1, step 3). Prepare the solution as follows.
Note: Each component must be added in the proper sequence
1. Add complete growth medium (2 mL for a 60 mm plate and 3 mL for a 100 mm plate) warmed to 37°C.
2. Add plasmid DNA, 10 ng to 10 µg. Gently mix.
3. Add polybrene to a final concentration of 5 µg to 10 µg per mL. Gently mix.
Remove medium from plate and add DNA-Medium-Polybrene solution to cells. Incubate cells at 37°C for 6 to 20 hours with occasional gentle rocking approximately every 1.5 hours for the first 6 hours.
Remove DNA-Medium-Polybrene solution and gently overlay cells with DMSO shock solution (15% DMSO in 1X HBSS): 3 mL per 60 mm dish and 4 mL per 100 mm plate. Manually rock the dish for 10 seconds to evenly distribute the solution, and then incubate the cells for exactly 4 minutes at 37°C.
Immediately remove the DMSO shock solution and gently rinse the cells twice with complete growth medium, 5 mL per wash per 60 mm dish, 10 mL per wash per 100 mm dish.
Add complete growth medium to the cells.
For Stable transformants, remove the growth medium and split the cells 1:5 into selection medium.
For transient expression, remove the growth medium and add fresh growth medium. Harvest cells and/or medium after 24 to 72 hours.

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