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1、Anewprotocolforgenotyping李广瑞(LiGuangrui)UniversityofTokyoE-mail:2012-5-251. Digestapproximately2mmmousetail(maybestoredat20C)with300yLbufferina1.5-mLEppendtourbfeat55-60Covernight.2. Nextmorning,labelonesetofmicrofugetubeswithmouseeartagnumbers.3. Centrifugeatfullspeed(20,0)30fo严5min.4. Transfer100u
2、Lsupernatanttoacleantubewithidenticalnumber.5. Add300uL100%ethanolintoeachtube,mixcompletely.6. Centrifugeatfullspeed(20,000foF10min,thenpourouttheethanol.7. WashtheDNApelletsbyadding300uL70%ethanolintothetubesverygenflushtheDNApellets)andcentrifugeatfullspeged)(for20,0500miFn,thenpourout70%ethanol.
3、8. Turntubesupsidedownonpapertoweltoair-drytheDNAfor1-2min(dorDNAmaybedifficulttoresuspend)9. DissolvetheDNApelletswith20-50uLTEbuffer.10. PolymeraseChainReaction(PCR)FORMULAappropriateamount12.5uL10-100pmol10-100pmol0.1uL(12.5units/uL)0.1-2ugDDW10FBufferVer.2Primer-ForwardPrimer-ReverseDNAPolymeras
4、eDNATemplate25uL11. ElectrophoresisGelCheckwhat%gelneeded*:AgaroseeffectiveResolDNA(kb)0.5%30to10.7%12to0.81%10to0.51.2%7to0.41.5%3to0.2*fromcurrentprotocolinNeuroscienceLysisBuffer0.5MEDTA50ml5MNaCl10ml1MTrispH7.45ml10%SDS50mlProteinaseK(10mg/ml)10mMTrispH7.320mMCaC2l50%glycerolTEbuffer10mMTris-HCl0.2mMNa2EDTApH7.5
