Day

1. For each 6-cm (or 10-cm) dish to be transfected, place 0.3 mL (or 0.6 mL) of 2X HBS into a sterile 4-mL polypropylene tube.

3. Add 18 |L (32 |L) of filter-sterilized 2 M CaCl2 dropwise while gently mixing (i.e., tapping the tube) or slowly vortexing.

4. Flick or slowly vortex tube for 20 s.

5. Let stand at room temperature for 45 min to allow a precipitate to form.

6. Remove media from each dish to be transfected by thorough aspiration. Alternatively, this media can be saved and replaced later.

7. Add DNA precipitate to the center of the dish, and rock gently by hand. Incubate at room temperature in the tissue-culture hood for 20 min (rock gently after 10 min).

8. Add 5 mL CEF media (or replace original medium), and incubate at 37°C for 3-4 h.

9. Remove media, and aspirate well. Carefully add 1 mL (2.5 mL) of sterile 15% (v/v) glycerol in HBS, and rock gently. Incubate at room temperature for exactly 90 s.

10. Wash with 5 mL of CEF media, rocking gently to mix.

11. Remove media, and repeat the same wash once or twice.

12. Remove final wash, add fresh CEF media, and incubate at 37°C.

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