The follicles from each culture were homogenized in a buffer containing 100 mM NaCl, 4 mM EDTA, 50 mM Tris-HCl, 0.5% SDS, pH 8, and proteinase K (100 ig/ml) at 55°C for 4 h to facilitate membrane and protein disruption. After incubation, samples were cooled for 30 min on ice in 1 M potassium acetate and 50% chloroform to initiate protein precipitation and then centrifuged at 9000 X g for 8 min at 4°C. Supernatants were then precipitated for 30 min in 2.5 volumes of ethanol at —70°C and centrifuged for 20 min at 5000 X g at 4°C.
Finally, samples were extracted in 70% ethanol and resuspended in water. DNA content was measured by reading the absorbance at 260 nm and then incubated for 1 h with RNase (10 ig/ml) at 37°C. DNA samples (4 ig) were electrophoretically separated on 1.9% agarose gels containing ethidium bromide (0.4 ig/ml) in Tris-borate-EDTA buffer. Within each agarose gel, equal amounts of DNA were loaded into each well. DNA was visualized in an ultraviolet (302 nm) transilluminator and photographed with a Polaroid camera system. Densitometric analysis of low-molecular-weight (<15 kb) DNA was performed with an Image Scanner (Genius) by the software program Scion Image for Windows (Scion Corporation, Worman’s Mill, CT).