Posted on June 3rd, 2016 by UNM CC
M. Fero — 6/01
(after C Gorman, M. Calos)
- 2x 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
(The volumes can be scaled up as necessary)
- Let all solutions equilibrate to room temperature.
- Feed cells on a 10 cm dish with 7 mL fresh media. They should not be more than 50% confluent.
- Add 0.5 mL of 2x HEPES to a conical tube.
- 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) H2O (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.
- Change the media the following day.