BIO-RAD电转化仪使用教程(自制)

BIO-RADGenePulserXcellElectroporationSystemBIO-RAD电转化仪使用教程（自制）cexoihtydx20110902一电转仪示意图Figure1connectingtheshockpadtotheGenePulserXcellmainunit.Figure2Shockpodwithcuvette.Figure3GenePulserXcellfrontpanel.二电转仪主界面在主界面中，我们经常会用到：4.Pre-setprotocols和5.UserprotocolsPre-setprotocols(预置方案)中，有Bacterial,Fungal和Mammalian三种预置方案。下面简单介绍一下Bacterial中E.coli和Fungal中Pichiapastoris的电转化方案和注意事项。三ElectroporationofBacterialCells(E.coli)1制备电转化感受态细胞1).Inoculate5mlofafreshovernightE.colicultureinto500mlofL-brothina2.8LFernbachflask.2).Growthecellsat37°Cshakingat300rpmtoanOD600ofapproximately0.5–0.7.Thebestresultsareobtainedwithcellsthatareharvestedatearly-tomid-logphase;theappropriatecelldensitydependsonthestrainandgrowthconditionsbutshouldbeabout4–5x107cells/ml.3).Chillthecellsonicefor~20min.Forallsubsequentsteps,keepthecellsascloseto0°Caspossi-ble(inanice/waterbath)andchillallcontainersinicebeforeaddingcells.Transferthecellstoasterile,cold500mlcentrifugebottleandcentrifugeat4000xgfor15minutesat4°C.4).Carefullypouroffanddiscardthesupernatant.Itisbettertosacrificeyieldbypouringoffafewcellsthantoleaveanysupernatantbehind.5).Gentlyresuspendthepelletin500mlofice-cold10%glycerol.Centrifugeat4000xgfor15minutesat4°C;carefullypouroffanddiscardthesupernatant.6).Resuspendthepelletin250mlofice-cold10%glycerol.Centrifugeat4000xgfor15minutesat4°C;carefullypouroffanddiscardthesupernatant.7).Resuspendthepelletin~20mlofice-cold10%glycerol.Transfertoa30mlsterileOakridgetube.Centrifugeat4000xgfor15minutesat4°C;carefullypouroffanddiscardthesupernatant.8).Resuspendthecellpelletinafinalvolumeof1–2mlofice-cold10%glycerol.Thecellconcentrationshouldbeabout1–3x1010cells/ml.9).Thissuspensionmaybefrozeninaliquotsondryiceandstoredat-70°C.Thecellsarestableforatleast6monthsundertheseconditions.2电转化转化参数：Pre-setprotocols(E.coli)Voltage(V)1800Capacitance(µF)25Resistance(ohm)200Cuvette(mm)11).Thawthecellsonice.Foreachsampletobeelectroporated:placea1.5mlmicrofugetubeonice,placeeithera0.1or0.2cmelectroporationcuvetteonice,andplacea17x100mmtubewith1mlofSOCatroomtemperature.2).Toacold,1.5mlpolypropylenemicrofugetube,add20µlofcellsuspensionifelectroporatingin0.1cmcuvettes,or20–40µlofcellsuspensionifelectroporatingin0.2cmcuvettes.Add1to2µlofDNA(DNAshouldbeinalowionicstrengthbuffersuchaswaterorTE).Mixwellandincubateonicefor~1minute.(Note:itisbesttomixtheplasmidsandcellsinamicrofugetubesincethenarrowgapofthecuvettespreventsuniformmixing.)3).FromtheHomescreenonGenePulserXcellopenthePre-setProtocolsscreen,thentheBacterialProtocolscreen(press4,thenEntertwice).Whenusingthe0.1cmcuvettes,pressEntertoopenE.coli,1mmcuvetteProtocolDetailscreen.Whenusingthe0.2cmcuvettes,press2thenEnter,or3thenEnter,toselecttheProtocolDetailscreensforE.colitopulseat2.5or3.0kV,respectively.4).TransferthemixtureofcellsandDNAtoacoldelectroporationcuvetteandtapthesuspensiontothebottom.PlacethecuvetteintheShockPod.Pushthechamberliddowntoclose.5).Pulseonce.6).Removethecuvettefromthechamberandimmediatelyadd1mlofSOCmediumtothecuvette.QuicklybutgentlyresuspendthecellswithaPasteurpipette.(TheperiodbetweenapplyingthepulseandtransferringthecellstooutgrowthmediumiscrucialforrecoveringE.colitransformants(Doweretal.,1988).Delayingthistransferbyeven1minutecausesa3-folddropintransformation.Thisdeclinecontinuestoa20-folddropby10minutes.)7).Transferthecellsuspensiontoa17x100mmpolypropylenetubeandincubateat37°Cfor1hour,shakingat225rpm.8).Checkandrecordthepulseparameters.Thetimeconstantshouldbecloseto5milliseconds.Thefieldstrengthcanbecalculatedasactualvolts(kV)/cuvettegap(cm).9).PlateonLBplateswithantibiotic.3溶液和试剂1).L-Broth:10gTryptonepeptone,5gYeastextract,5gNaCl;dissolvein1.0Lwater.Autoclave.2).LBagarplateswithselectiveantibiotic:prepareLbrothasabove,adding15gofagarperliter.Autoclave.Coolto55–60°Candaddantibiotic.Pour12–15mlper100mmplate.3).10%(v/v)Glycerol:12.6gglycerol(density=1.26g/cc)in90mlofwater.Autoclaveorfiltersterilize.4).TE:10mMTris-HClpH8.0,1mMEDTA.5).SOB:2.0gTryptonepeptone,0.5gYeastextract,0.2ml5MNaCl,0.25ml1MKCl;dissolvein90mlwater.AdjustpHto7.0.Bringvolumeto100ml.Autoclave.Add1.0mlsterile1MMgCl2and1.0mlsterile1MMgSO4.6).SOC:to100mlSOB,add2.0mlsterile1Mglucose(sterilizebyfiltration).4注意事项1).细菌电转化电压一般默认为1.8KV即可，某些细菌可能会需要更高的电压，可以参考电转化仪器厂商的相关资料以及文献报道。2).10ul过夜连接产物，如果想直接用连接产物进行电转化，则电转化的连接产物加入体积不能超过4ul,不然很容易产生电火花。如果想将10ul连接产物全部用于电转化，以提高转化效率，可进行一次DNA浓缩（乙醇沉淀）。方法为：10ul连接产物，加15ul无菌水H2O，2.5ul3MNaAc及60-70ul无水乙醇，混匀，-20℃冻存30min,4℃离心，去上清，再用70%乙醇沉淀DNA，挥干乙醇，溶于5ulddH2O中，取1-2ul用于电转化。四ElectroporationofFungalCells(Pichiapastoris)1制备电转化感受态细胞1).Inoculate500mlofYPDina2.8LFernbachflaskwithanaliquotfromafreshovernightcultureofP.pastoris.ThedoublingtimeofP.pastorisisapproximately2hrsat30°C.2).Incubateat30°Covernight,shakingat300rpm,toadensityof~7x107cells/ml[OD600(1:10dil)=0.24–0.30].3).Decantthecellsintoasterile500mlcentrifugebottleandpelletthecellsbycentrifugationat3000xgfor5minat4°C.4).Carefullypouroffanddiscardthesupernatant.5).Add100mlofsterile,YPD/HEPEStoeachofthebottlesandvortextoresuspendthecellpellets;add2.5mlof1MDTT;mixg