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).
免责声明 本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘”。违反上述声明者,本网将追究其相关法律责任。