基因敲除详细步骤

1ThefollowingprotocolstakeMLCK(myosinlightchainkinase)asanexample.Generalsteps:1.BACextraction(ItisnecessaryforustoidentifytheBACbyPCR)2.TransformBACtoEL350(Cm+)3.Retrieving(Cm+Amp+)4.Targeting1stloxP(Amp+Amp+andK+)5.TransformMLCK1stloxPtoEL350togetpurifyMLCK1stloxP(Amp+andK+)6.MLCK1stloxPpopout(Amp+andK+AmP+)7.TransformMLCK1stloxPpopouttoEL250(Amp+)8.Targeting2ndloxP(Amp+Amp+andK+)9.TransformMLCK2ndloxPtoDH-5αorXL1-Blue(Amp+andK+)10.Linearization1.BACextractionSolutionI:Tris.Cl0.025MEDTA0.01MGlucose0.05MpH8.0SolutionII:SDS1%NaOH0.2Mfreshprepared(1Volume2%SDS+1Volume0.4MNaOH)SolutionIII:(120ml5MKAc+23mlHAc+57mlH2O)/200mlProtocol:21.Harvest50mlbacterialcells(O/N)bycentrifugationat9,000r/mfor10min,pouroffthesupernatantclearly.2.Add5mlice-coldSolutionI,mix.3.Add10mlSolutionII,invertseveraltimesgently.4.Add7.5mlSolutionIII,invertseveraltimesgently.5.Centrifugeat9,000r/minfor10minat4℃.Removethesupernatanttoafreshtube.6.Add0.6volumeofisopropanol7.Centrifugeat9,000r/minfor10minat4℃.Removesupernatant.8.DissolveDNApelletin400ulTE,add20ul10mg/mlRNase,55℃,20-30min.9.AddequalvolumeofPhenol/chloroform(1:1),mixandcentrifugeat12,500rpmfor5minatRT.(Fromthisstep,1.5mltubecanbeused)10.Transfersupernatanttoafreshtube,addequalvolumeofchloroform,mixandcentrifugeat12,500rpmfor10minatRT11.Add0.1volumeof3MNaAc(pH5.3)and2volumeofethanol(storedat-20℃).Mixandcentrifugeat12,500rpmfor10minat4℃.12.DissolveDNAwith50ulpH7.4MilliQH2O.(TENSisn’tgoodforBACextraction)2.TransformBACintoEL350(Cm+)1.PickupasinglecolonyofEL350to3mlLB,growat32℃O/N(12-16h)2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2-3htoOD600=0.5Fromthisstep,alloniceorin4℃3.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min4.Spinat3,500rpmfor6min,resuspendcellwith1.5mlpH7.4MilliQH2O.5.Spin,washtwicemore.6.Removesupernatant,addabout50ulpH7.4MilliQH2O.7.Add10ulBACto50ulcompetentcells,pipettethemtoanelectroporationcup(0.1cMgap).8.1.75kV,25uF,200ohms.9.Add1mlSOCtoeachcurvetteandincubateat32℃for1h.10.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernantandplatecells(Cm+).33.Retrieving(Cm+Amp+)Retrievalplasmidconstruction1.PCRamplifytwo500bphomologousarms（BACastemplate）2.InsertthesetwofragmentstoTvector3.NotI,HindIIIcuttingandSpeIHindIIIcutting500bpfragmentsligatedwithNotI,SpeIcuttingPL2534.Transform,identifythepositivecolones.5.CuttheretrievalplasmidwithHindIII,purifytheproduct.Transformation1.PickupasinglecolonyofBAC-EL350to3mlLB,growat32℃O/N(12-16h)2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2-3htoOD600=0.53.InduceBAC-EL350in42℃waterbathbyshakingfor15min.Fromthisstep,alloniceorin4℃4.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min5.Spinat3,500rpmfor6min,resuspendcellswith1.5mlpH7.4MilliQ6.Spin,washtwicemore.7.Removesupernatant,addabout50μlpH7.4MilliQ8.Add200-500nglinearretrievalplasmidto50ulBAC-EL350,pipettethemtoanelectroporationcup(0.1cMgap).9.1.75kV,25uF,200ohms.10.Add1mlSOCtoeachcurvetteandincubateat32℃for1h.11.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernatantandplatecells(Amp+).4242526272829MLCK-BACCm242526272829PL253retrievingvectorCmMLCK-BACCm+Amp+Amp+RetrievingMLCK-PL253MLCK-PL253digestionmapEL350,42℃4.Targeting1stloxP(Amp+Amp+andK+)Mini-targetingvectorconstruction1.PCRamplify80bphomologousarmsloxPfloxedNeocassette(1stloxP)fromPL452.2.Ligate1stloxPwithTvector(IfthePCRproductquantityisenoughforlateruse,thisprocedureisnotnecessary)3.Cut1stloxP–Ttoget1stloxPfragment,purifytheproduct.Note:Ifyouusetheligationmethodtoconstructthemini-targetingvector,two~500bphomologousarmsareligatedtothefloxedNeocassetteexcisedfromPL452withEcoRIandBamHIandtoPL253thatislinearizedbyNotIandSalI.Formoredetail,plssee,Lee,E.C.etal.AhighlyefficientEscherichiacoli-basedchromosomeengineeringsystemadaptedforrecombinogenictargetingandsubcloningofBACDNA.Genomics73,56-65(2001)Transformation1.PickupasinglecolonyofMLCK-PL253-EL350to3mlLB,growat32℃O/N(12-16h)2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2-3htoOD600=0.53.InduceMLCK-PL253-EL350in42℃waterbathbyshakingfor15min.Fromthisstep,alloniceorin4℃54.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min5.Spinat3500rpm6min,resuspendcellwith1.5mlpH7.4MilliQ6.Spin,washtwicemore.7.Removesupernatant,addabout50μlpH7.4MilliQ8.Add200ng1stloxPfragmentto50ulMLCK-PL253-EL350,pipettethemtoanelectroporationcup(0.1cMgap).9.1.75kV,25uF,200ohms.10.Add1mlSOCtoeachcurvetteandincubateat32℃for1h.11.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernatantandplatecells(Amp+andK+).252627MLCK-PL253MLCK-PL2531stLoxPNeoAmp+Amp+K+Amp+Amp+LoxpfloxedNeocassetteTargeting1stLoxPMLCKPL253digestionmapEcoRIMLCK-PL2531stLoxPdigestionmapHindIIIEcoRIEL350,42℃5.TransformMLCK1stloxPtoEL350togetpurifiedMLCK1stloxP(Amp+andK+)Note:EL350containsmultiplecopiesofplasmids,sotherewillbebothrecombinantandunrecombinantplasmidsinEL350.6.1stloxPpopout(Amp+andK+Amp+)61.PickupasinglecolonyofEL350to3mlLB,growat32℃O/N(12-16h)2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2htoOD600≈0.4.3.InduceEL350with1mg/mlArabinoseat32℃for1h.Fromthisstep,alloniceorin4℃4.Transfer6mlcellsto15mlcentrifugetube,