分子生物学常用实验技术

分子细胞遗传学常用技术2007年8月1分子细胞遗传学常用实验技术GeneralLaboratoryGuidelinesA.Beforestarted:Beforeyouenterthelaboratory,makesureyouhavereadandfullyunderstandthelaboratorysafetymanual.Pleasereadthelaboratorymanualbelowandfollowtheguidelinesofeachexperiment.KeepinmindthatalltheguidelinesinthismanualareINGENERALbutnotforeverydetailsteps.Makesureyouhavefullyunderstoodeachstepintheprotocolanditsreason.Pleaseaskyoursupervisorifyouhaveanythingunclear.B.Solutions-Ifyouuseasolutionandthevolumeislow(lessthan500ml),notifythepersonwhomakesit.Donotwaituntilthereisalmostnoneleft.Ifyouanticipateusingalotofaspecificsolution,youmaywanttomakesomeforyourself.Makesureallsolutionsarelabeledwithyourinitials,dateandthechemicalnameandconcentration.C.Pipettipsandmicrofugetubes-Afteryouuseaboxofpipettips,refillitandplaceitontheshelf.Whenseveralaccumulatetechnicianwillautoclavethemandreturntothedrawer.Keepyourplasticboxofmicrofugetubes.Youcanjustrefillyourboxasnecessary.Don’taccumulatemanypipetboxeswithoutrefillingthemorotherswon’thaveanytouse.D.Autoclave-Donotautoclavewasteitemswithsterileitems.E.Orders-Ifyouseethatsomethingisrunningloworifyouanticipateusingalotofanitem,notifytheresponsiblepersonandhewillplacetheorder.Donotwaituntiltheitemisgoneoralmostgone-givetheorderinadvance.F.LaminarFlowHood-Makesureyousprayitdownwith70%ethanolbeforeandafteryouuseit.Ifthehazardouswasteisfilled-autoclaveit.Donotletitoverfill-thewasteshouldbedisposedofweekly.G.Dishes-Wehaveaverysmallsinksowashyourglasswareeveryday.Washwithsoapandwater.Rinsewithdistilledwater.Whenyouritemshavedried,putthemaway.Don’tletitemsaccumulatenearthesink.Thesamegoesforyourbench-cleanupaftereveryuse.H.Equipment-Ifyouhaveaproblemwithanyequipment,notifytheresponsiblepersonimmediately.Beforeusinganyequipment,askfordirectionsorreadthemanualfirst.I.Pipets-Donotsetpipetsovertheirspecifiedrange.Pipettingsolutionupanddownslowly.Takegreatcarewhenpipetschemicalslikechloroform.Referto分子细胞遗传学常用技术2007年8月2thefactorywebsiteforinstructionsonhowtofixbrokenpipetsortheinstructionbooklet.P-20:2-20ulP-200:50-200ulP-1000:100-1000ulJ.RefrigeratorandFreezer-Alwaysmakesurethedoorstotherefrigeratorsandfreezersareclosedwhenyouarefinishedwiththem.K.NylonMembranes-AllSouthernfiltersshouldbewrappedinsaranwrapandstoredinasealedziplockbagtopreventmoistureloss.Makesurethebagislabeled“Stripped”iftheyhavebeenusedbeforeandarereadyforre-probing.L.Opensampleorregents’tubes:Alwaysdoashortspinbeforeyouopenanysampleandregenttubestoeliminateanypossiblecontamination.M.TransferhighconcentrationDNAsamples:DuringDNAextractionandafterwardpurification,makesureyourcutofftheendofthepipettips.ThesharpendoftipswillbreaktheDNAmolecular.分子细胞遗传学常用技术2007年8月3第一部分常用试剂、培养基的配制0.5MEDTApH8.0EDTA2Na2H2O186.1gNaOH～20gH2O→1L3MNaAcpH5.2高压灭菌NaAc3H2O408.3g醋酸调节pH至5.2ddH2O800ml1000ml5MNaClNaCl146.1gddH2O→500ml10%SDSpH7.2SDS20gHClafewdrops200ml1MTris-HClpH8.0Trisbase121.18gHCl～42mlddH2O→1000mlTEpH8.0Tris-HCl(pH8.0)10mlEDTA2mlddH2O→1000ml分子细胞遗传学常用技术2007年8月4TE0.1pH8.0Tris-HCl(pH8.0)10mlEDTA200μlddH2O→1000ml50×TAETrisbase242g冰醋酸57.1ml0.5MEDTA(pH8.0)100mlddH2O→1000ml10×TBETrisbase108g硼酸55g0.5MEDTA40mlddH2O→1000ml1MKCl(M.W.74.5)KCl7.45gddH2O→100mlGel-loadingBuffers(foragarosegel)溴酚蓝（0.25%）100mg蔗糖20gddH2O→50mlE.B.10mg/mlE.B.1gddH2O100mlHCl(1N11.6M/L)分子细胞遗传学常用技术2007年8月536.5%HCl86.2mlH2O913.8ml1M葡萄糖（M.W.180）葡萄糖18gddH2O→100ml过滤除菌，0.22μm1MMgCl2（M.W.203.3）注意：吸湿性强，空气中迅速潮解，请迅速配制MgCl26H2O20.32gddH2O→100ml灭菌1MMgSO4（M.W.246.5）MgSO424.64gddH2O→100ml不易溶解，需要使用搅拌器灭菌IPTG储存液（M.W.238.3）2g溶解于8mlddH2O中，通过0.22μm过滤器，过滤除菌，调整至10ml。分装，保存于-20℃。X-gal储存液20mg/ml二甲基甲酰胺于玻璃瓶或聚丙烯管中，用铝箔包裹严密，不需要过滤除菌，保存在-20℃。抗生素抗生素浓度保存条件氨苄青霉素50mg/ml(溶于水)-20℃卡那霉素10mg/ml(溶于水)-20℃氯霉素50mg/ml(溶于乙醇)-20℃以水为溶剂的抗生素储存液应用0.22μm过滤器，过滤除菌用乙醇溶解的抗生素溶液无需除菌处理，所有的抗生素贮存液都应放于不透光的容器中保存20×SSCNaCl3M(58.44×3=175.32)柠檬酸纳0.3M(294.1×0.3=88.23)分子细胞遗传学常用技术2007年8月6用1NHCl调节pH（7.0）10×PBSNaCl1.3M(58.44×1.3=75.97)Na2HPO40.07M(141.96×0.07=9.94)NaH2PO40.03M(120×0.03=3.6)(NaH2PO4H2O137.99×0.03=4.14)Na2HPO42H2O0.07M(358.14×0.07=25.07g)NaH2PO42H2O0.03M(156.01×0.03=4.68g)柠檬酸buffer0.01MpH4.5Na3C6H5O72H2O1.47gC6H8O71.05gddH2O→500ml鲑鱼精DNA制备(salmonspermDNA)①Dissolve1gDNA(anyfishspermDNA)in100ml0.4MNaOHovernight②Boil30mins(片断长度为200-1000bp)③Chillneutulizewithconcentratedglacialaceticacid(～2ml)④Spinatdehriifany⑤Add2VEtOHincubate-20℃for≥1h⑥PelletDNA-washwith70%EtOHdry⑦Resuspendin100mlTE=10mg/mlSephadexG-50AddSephadexG-50(medium)todistilledsterilewater.WashtheswollenresinwithH2Oseveraltimestoremovesolubledextran,whichcancreateproblemsbyprecipitatingduringethanolprecipitation.Finally,equilibratetheresininTE(pH7.6),autoclave(101b/sqinfor15min)andstoreatroomtemperature.培养基的配制SOCSOB10mlGlucose(1M)200μlMgSO4(1M)100μlMgCl2(1M)100μlSOB分子细胞遗传学常用技术2007年8月7Bactotryphone2gYeastextract0.5g5MNaCl0.2ml1MKCl0.25ml5NNaOH～0.02mlddH2O→100ml高压灭菌LB培养基tryphone10gYeastextract5gNaCl10gddH2O→1000ml5NNaOH～0.2ml固体LB培养基1L向液体培养基中加入1.5%（1000ml→15g）的琼脂,并且加入：X-gal（2%）1000μlIPTG（20%）100μl2×YTtryphone16gYeastextract10gNaCl5g如果需要用1NNaOH(～1ml)调节pH至7.0ddH2O→1000ml分子细胞遗传学常用技术2007年8月8第二部分常用技术植物基因组DNA的提取①