DNA Extraction from Cheek Cells
来自 : 蚂蚁淘
ExtractionfromCheekCellsDNASamplePreparation
Cheekcellsareobtainedbyrinsingthemouthwith25mlsofanycommercialmouthwashsolutionavailableforabout30secthefirstthinginthemorning.Itisimportantnottobrushyourteethbeforecollectingthespecimen.Themouthwashisthendiscardedintoasterileconicaltubeandsenttothelaboratory.
ASaltingOutProcedureforDNAextractionfromCheekCells
- Tubecontainingmouthwashiscentrifugedat4000rpmfor15minutes.
- SupernatantisremovedcautiouslybymeansofasterilePipette,withoutdisturbingthepelletatthebottom.
- Pelletiswashedwith25mlTEbuffer,bymeansofcentrifigationat4000rpmfor10minutes.
- Repeatsteps2and3.
- ThepelletisresUSPendedin1mlSEbuffercontaining1μlproteinaseK(1mg/ml)and1%SDS.
- Incubateat370Covernightinawaterbath.
- Add1mlSEbuffer
- Add500μl6MNaCl
- Add2.5mlchloroform
- Mixfor60mininanover-the-toprotator.
- Vortexfor20sec.
- Centrifugefor10minutesat2000rpm,andsettheslowestbreakingforce
- Transferthesupernatanttoacleantube.
- Addanequalvolumeofisopropan-2-oltothesupernatanttoprecipitatetheDNA.
- IfDNAisvisIBLegotostep16andifnotgotostep17.
- IfDNAisvisible,spoolouttheDNAandplaceinmicrofugetubefilledwith1mlof75%ethanol(gotostep18)
- IfDNAisNOTvisiblecentrifugethetubefor4minutesat11,000ganddiscardtheisopropanol.Add1mlof75%ethanol.
- Centrifugefor4minutesat11,000ganddiscardtheethanoltakingcarenottolosetheDNA.
- Addethanolandrepeatcentrifugation2times.Discardethanolafterlastcentrifugation.
- Evaporateethanolbyplacingmicrofugetubeinoven(max60ºC)forabout20-30mins.
- Add100μlofTEbuffertorehydrateDNA,andmixovernightonanoverthetoprotator.
Fig.1.AmplificationofCTLA4genebyPCR.TracksmarkedCusingDNAextractedfromcheekcells,therestDNAextractedfromperipheralbloodleucocytes(WB).
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