Chromosome and nuclei isolation with the MgSO4 procedure.

摘要: Publisher Summary MgSO 4 isolation procedure employs Mg 2+ to stabilize chromosomes in suspension. The procedures are used successfully produce high-resolution flow karyotypes from a variety of cell types. Mitotic cells swollen hypotonic buffer containing this stabilizing agent, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic (HEPES) acid as (pH 8.0) and dithiothreitol (DTT) reducing agent. This combination effectively reduces the number chromosome clumps preparation. If propidium iodide (PI) is be DNA-specific stain, RNase also added buffer. It essential that growth medium completely removed mitotic pellet before addition ensures sufficient hypotonicity for swelling. Chromosomes released form monodispersed suspension with less rigorous mechanical shearing thus damage. stained fluorochromes. stains equilibrate analysis; chromomycin binding considered complete after 1–2 hours. Chromosome resolution greatly improved by 10 mM sodium citrate 25 sulfite at least 15 minutes analysis. prepared method stored 4°C 24–48 hours