Materials2x HEPES: 8 g NaCl, 0.105g NaHPO4 , 6.5 g HEPES, H2O to 500 mL, pH 6.95 to 7.10 (try a range) 2M CaCl2 : store in aliquots at -20°C DNA 4 mg/mL Procedure(The volumes can be scaled up as necessary) 1) Let all solutions equilibrate to room temperature.
2) Feed cells on a 10 cm dish with 7 mL fresh media. They should not be more than 50% confluent.
3) Add 0.5 mL of 2x HEPES to a conical tube.
4) To a separate tube add 61 µL of 2M CaCl2 , plus 4 - 10 µg (1 - 2.5 µL) DNA, and q.s. to 0.5 mL by adding (438 µL) H2 O (or try TE pH8.0 ). Mix with a pipet and transfer dropwise to the 2x HEPES solution. Mix and then immediately sprinkle on top of the cultured cells. Swirl gently and return to the incubator overnight.
5) Change the media the following day.
