Dimethyl Sulfoxide → Tricalcium Phosphate

Participants

Reaction Steps

Seventy-five micrograms (75 μg) of DNA were dissolved in 1.35 ml water in a 50 ml conical tube. One hundred and fifty microliters (150 μl) of 2.5 M CaCl2 were added, and this DNA-calcium mixture was added dropwise to 1.5 ml of 2× HeBES (HEPES buffered saline) in a 50 ml conical tube. The HeBES was bubbled with a pipettor while adding the DNA-calcium mixture with a Pasteur pipette. The mixture was mixed by vortex for 5 seconds and incubated at room temperature for 20 minutes. One milliliter (1 ml) of the DNA-calcium mixture was distributed evenly over each culture dish of adherent cells, grown and prepared for transfection in F12 medium as described above, and the cultures then incubated at 37° C. for four hours. After incubation, the plates were aspirated, and 2 ml of 10% dimethylsulfoxide (DMSO) in F12 medium was added to each plate (DMSO shock treatment). The DMSO shock continued for one minute after which the DMSO was diluted by the addition of 5 ml of PBS (phosphate buffered saline) to each plate. The plates were aspirated and washed two more times in PBS. Ten (10) ml of αMEM/5% FBS/HT was added, and the plates were incubated at 37° C. overnight.

Provenance & Evidence