ThisproceduredescribesaconvenientmethodfortheisolationofcrudenuclearpelletsfromN.crassa.Themethod,anadaptationoftheonedevelopedbyHautalaetal((1977JBact130:704-713)utilizesaBraunhomogenizertodisruptthecells.Themainadvantagesofthetechniquearethatthecellsneednotbefroen,largeamountsofmaterialcanbehandled,thehomogenizationisfastandeasilycontrolled,feweromni-mixstepsandshotertimesarerequiredtoreleasethenuclei,yieldsarecomparable(75%)tothoseobtainedusingthefrenchpressurecell,andlowerconcentrationsofFicollwillstABIlizethenuclei.ThecrudenuclearpelletsareusedtoprepareDNAandpurenuclei.

Germinatingconidia(14hrs)areharvestedbyfiltrationandrinsed.TheBraunhomogenizerdisruptscellsviahighspeedshaking(4000rpm)withglassbeads.Typically90gwetweightofcellsareusedineachisolation.The90garedistributedamongfour75mlglasshomogenizerbottles.Eachbottlecontains50gacidwashedglassbeads(0.45-0.50mm),10-15gcellsand11mlisolationbufferA(Hautalaetal1977).Theisolationbuffer,however,containsonly5%ficoll400.ThecellsarekeptcoldduringthehomogenizationbyajacketfedwithsIphonedCO₂.Thecellsarehomogenizedin30secpulsesfollowedby30secrests.Table1showsthatoptimumyieldswithoutlysingnucleiareobtainedusing90sectotalhomogenizationtime.Theyieldat120secisthesamebut20%ofthenucleihavelysed.Thehomogenatesplusbeadsfromthefourbottlesarecombinedinabeakerandallowedtosettlefortwominutes.Thehomogenateisthendecantedfromthebeads.Thebeadsarerinsedthreeorfourtimeswith50mlofisolationbufferandaresavedforreuse.Thehomegenateandrinsesarecombinedandtheirvolumeadjustedto300ml.Themixtureisthenomni-mixedfor10-15minutesatasettingof6.0,Thesolutionisthencentrifugedat700xginlargeplasticcentrifugebottlesfor10minutes.Thedecantedsupernatantissaved.ThepelletisresUSPendedwithasyringeinisolationbufferthevolumeadjustedto300mlandomni-mixedasecondtimeusingthesameconditions.Thesolutioniscentrifugedandthesecondspiniscombinedwiththefirstsupernatant.Thecrudenuclearpelletisobtainedbycentrifugingthecombinedsupernatantsat9000xgfor50minutes.Weroutinelyobtainyieldsof65-75%basedonDNAcontentusingthismethod.SeeTable11.Theentireprocedurerequiresaboutfourhours.itispossIBLetohandle180gofcellsbyrunningtwohomogenizations.Whilethefirsthomogenateisomni-mixedandcentrifuging,thesecondhomogenatemaybestartedintheomni-mixer.Byoverlappingthecentrifugeandomni-mixtimesinthismannerandcombiningallthesupernatantstospindownthecrudenuclearpelletwecanhandle180ginfourhoursand360gconvenientlyinaday.(SupportedbyGrantGM-23367fromtheNationalInstitutesofHealth).---DepartmentofBiochemistryandTheDevelopmentalBIOLOGyProgram,---OhioStateUniversity,Columbus,Ohio43210.

 TABLE 1Efficiency of Cell Disruption with the Braun Homogenizer % yield and distribution of DNAtime (sec) of crude crude homogenization nuclear membrane nuclear---------------- pellet pellet supernatant ------ ------ ----------- 0 0 100 0 30 25 70 5 60 39 57 4 90 75 20 5 120 73 8 19 150 57 6 37 --------------------------------------------------------------------------homogenizations were performed in 30 sec pulses followed by 30 sec rests TABLE 11 Yield Comparisons of DNA Using Different Techniques Method of % yields based on DNA Homogenization whole cells crude nuclei pure nuclei---------------- ------------ ------------ -----------1. french pressure cell 100 70-80 25 (cell frozen)2. french pressure cell 100 65-72 22 (cell cold)3. Braun homogenizer 100 65-75 26 90 sec4. Hand shaking with 100 20 2-3 glass beads 10 min------------------------------------------------------------------------- DNA concentrations were measured by the diphenylamine method (Giles et al. 1965, Nature 206:93)