DROSOPHILARNAPREP(GoodmanLab) StockSolutions 3MNaOAc,pH5.2withHAc(MW=82) 6MGuanidineHydrochloridein0.1MNaOAc,pH5.2(MWGuHCl=95.54) 5.7MCesiumChloridein0.1MNaOAc,pH5.2(MWCsCl=168.37) Procedure 1.BrushembryosontoteflonpanwithddH2Oandfilteradultsonnitex. 2.RinsewellwithddH2Otoremoveyeast. 3.Scoopintobeaker. 4.Dechorionatewithchlorox:ddH2O(60:40);swirlfor2-3". 5.PourontonitexandwashthoroughlywithddH2O. 6.Allowembryostosettle5-10"andaspirateoffchorions. 7.Reapeatstep6withcleanddH2O. 8.Pourdechorionatedembryosontonitex,washwithmoreddH2Oanddrybriefly. 9.Weighembryos(usemax.3g/prep). 10.DouncebyhandusingtheApestle(1gembryos/10mlGuHCl-NaOAc). 11.Spin10"@10krpmin30mlCorextubestoremovecellulardebris. 12.Harvestthesupernatantandlayerontoa4mlCsClcushioninpolyallomertubes(13ml)fortheSW41rotor.Cfg.25krpm,18h@18oC. 13.TheRNAshouldappearasagelatinouspelletatthebottomofthetube.Removetheupper5-8mlCsCl-cellhomogenatebyaspirationorpipetting.PourofftheremainingsupernatantandkeepthetubeinvertedtoavoidbackwashingproteasesontheRNA!Usearazorbladetocutawaytheupperhalfofthetube. 14.CarefullyresUSPendRNAinDEP-H2O(orTE+0.2%SDS)andNaOAc/EtOHppt. N.B.TLA100.2preps:layer1.8mlGuHClextractona0.3mlCsClcushion;centrifuge4h,57krpm@20oC.
