作者: LOUISE B. PREER , GUY HAMILTON , JOHN R. PREER
DOI: 10.1111/J.1550-7408.1992.TB04448.X
关键词:
摘要: A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all unbroken cells and macronuclei approximately two thirds macronuclear fragments. Next higher 9,000 sediments micronuclei frees them small particulates soluble constituents. Advantage then taken fact that have lower density than do fragments in 45%-60% Percoll. Micronuclei float top during 24,000 g, while sediment. several cycles Percoll, micronuclei, although heavily contaminated with cytoplasmic components, are essentially free Micronuclear can be extracted suspension. The whole procedure very rapid about an hour separated. About 2 micrograms obtained 6 x 10(7) paramecia. We find there internal sequences gene wild type which eliminated when develop into macronuclei. They yield unique restriction Therefore purity preparations easily monitored by probing Southern blots enzyme-digested cloned gene. No differences found between d48 mutant.