MitochondrialDNAisisolatedbyamodificationofthemethodsdescribedbyWilsonandChourey(1984)andRadetzky(1990).

Cellculturesatfourdayspost-subculturearegroundat4degreesCelsiusinamortarandpestlewithsandandfivevolumes/gramfreshweightextractionbuffer(0.4Mmannitol,1mMethyleneglycol-bis[aminoethylether]N",N",N",N",-tetraaceticacid(EGTA),20mMN-[2-hydroxyethyl]piperazine-N"-[ethanesulfonicacid](HEPES),pH7.5,0.1%bovineserumalbumin,0.05%cysteine,10mMdiethyldithiocarbamicacid(DIECA),0.5%PolyclarAT(BDHChemicalsLtd.,England).

AfterfiltrationthroughtwolayersofMiracloththehomogenateiscentrifugedat150gfor10minutes.Thesupernatantisthencentrifugedthreetimesat3000gfor10minutestoseparatecellulardebris,nucleiandproplastidsfromthemitochondria.Mitochondriaarepelletedat10000gfor20minutes,resUSPendedinDNasebuffer(0.4Mmannitol,20mMHEPES,pH7.5,10mMmagnesiumchloride)andtreatedwithDNaseI(0.1mg/mL)at4degreesCelsiusforonehour.

AfterwashingthemitochondriawithDNaseinhibitingbuffer(100mMEGTA,0.4Mmannitol,20mMHEPES,pH7.5)mitochondriaarefurtherpurifiedbycentrifugationinadiscontinuousPercollgrADIent(45%,21%,14%Percoll)at15000gfor15minutes.Themitochondriathatbandarepooledanddilutedwithresuspensionbuffer(0.4Mmannitol,20mMHEPES,pH7.5,10mMEGTA,10mMDIECA)andcentrifugedseveraltimesat10000gfor10minutestoremovePercoll.

Thewashedandpelletedmitochondriaareresuspendedinlysisbuffer(0.1MTris.HCl,pH8.0,0.05MEDTA,0.1MNaCl,1%w/vsodiumdodecylsulfate,1%w/vsarcosyl)andincubatedat65degreesCelsiusfor30minutes.Organicmaterialisremovedbyadditionof5Mpotassiumacetatewithincubationonicefor20minutes.

Aftercentrifugationthesupernatantismixedwithanequalvolumeofisopropanol.PreciptatedDNAisdissolvedinTEbuffer(10mM/1mM).

DNAisfurtherpurifiedbyextractionwithphenol(bufferedwithTE),followedbytwoextractionswithchloroform:isoamylalcohol(24:1v/v),reprecipitatedanddissolvedinTEbuffer.RNAisremovedbyadditionofRNaseduringtherestrictionendonucleasedigestionofthemitochondrialDNA.

Notes:

1. DIECAmustbeaddedjustpriortouse.
2. Buffersandplasticwareareautoclavedpriortouse.
3. PerformDNAextractioninsterileflowhoodforbestresults.
4. Percollgradientstepscanbeeliminatedwithoutmuchlossinpurity.
5. RestrictiondigestionofLarixmitochondrialDNAfollowedbyagarosegelelectrophoresisresultedinacomplexbandingpatternwithbandsofvaryingstoichiometry.

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