基因敲除详细步骤.docx
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基因敲除详细步骤.docx
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基因敲除详细步骤
ThefollowingprotocolstakeMLCK(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.Linearization
1.BACextraction
SolutionI:
Tris.Cl0.025M
EDTA0.01M
Glucose0.05M
pH8.0
SolutionII:
SDS1%
NaOH0.2M
freshprepared(1Volume2%SDS+1Volume0.4MNaOH)
SolutionIII:
(120ml5MKAc+23mlHAc+57mlH2O)/200ml
Protocol:
1.Harvest50mlbacterialcells(O/N)bycentrifugationat9,000r/mfor10min,pouroffthesupernatantclearly.
2.Add5mlice-coldSolutionI,mix.
3.Add10mlSolutionII,invertseveraltimesgently.
4.Add7.5mlSolutionIII,invertseveraltimesgently.
5.Centrifugeat9,000r/minfor10minat4℃.Removethesupernatanttoafreshtube.
6.Add0.6volumeofisopropanol
7.Centrifugeat9,000r/minfor10minat4℃.Removesupernatant.
8.DissolveDNApelletin400ulTE¥,add20ul10mg/mlRNase,55℃,20-30min.
9.AddequalvolumeofPhenol/chloroform(1:
1),mixandcentrifugeat12,500rpmfor5minatRT.(Fromthisstep,1.5mltubecanbeused)
10.Transfersupernatanttoafreshtube,addequalvolumeofchloroform,mixandcentrifugeat12,500rpmfor10minatRT
11.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=0.5
Fromthisstep,alloniceorin4℃
3.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min
4.Spinat3,500rpmfor6min,resuspendcellwith1.5mlpH7.4MilliQH2O.
5.Spin,washtwicemore.¥
6.Removesupernatant,addabout50ulpH7.4MilliQH2O.
7.Add10ulBACto50ulcompetentcells,pipettethemtoanelectroporationcup(0.1cMgap).
8.1.75kV,25uF,200ohms.
9.Add1mlSOCtoeachcurvette¥andincubateat32℃for1h.
10.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernantandplatecells(Cm+).
3.Retrieving(Cm+Amp+)
Retrievalplasmidconstruction
1.PCRamplifytwo500bphomologousarms(BACastemplate)
2.InsertthesetwofragmentstoTvector
3.NotI,HindIIIcuttingandSpeIHindIIIcutting500bpfragmentsligatedwithNotI,SpeIcuttingPL253¥
4.Transform,identifythepositivecolones
5.CuttheretrievalplasmidwithHindIII,purifytheproduct.
Transformation
1.PickupasinglecolonyofBAC-EL350to3mlLB,growat32℃O/N(12-16h)
2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2-3htoOD600=0.5
3.InduceBAC-EL350in42℃waterbathbyshakingfor15min.
Fromthisstep,alloniceorin4℃
4.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min
5.Spinat3,500rpmfor6min,resuspendcellswith1.5mlpH7.4MilliQ
6.Spin,washtwicemore.
7.Removesupernatant,addabout50μlpH7.4MilliQ
8.Add200-500nglinearretrievalplasmidto50ulBAC-EL350,pipettethemtoanelectroporationcup(0.1cMgap).
9.1.75kV,25uF,200ohms.
10.Add1mlSOCtoeachcurvetteandincubateat32℃for1h.
11.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernatantandplatecells(Amp+).
4.Targeting1stloxP(Amp+Amp+andK+)¥sequence
Mini-targetingvectorconstruction
1.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)
Transformation
1.PickupasinglecolonyofMLCK-PL253-EL350to3mlLB,growat32℃O/N(12-16h)
2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2-3htoOD600=0.5
3.InduceMLCK-PL253-EL350in42℃waterbathbyshakingfor15min.
Fromthisstep,alloniceorin4℃
4.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min
5.Spinat3500rpm6min,resuspendcellwith1.5mlpH7.4MilliQ
6.Spin,washtwicemore.
7.Removesupernatant,addabout50μlpH7.4MilliQ
8.Add200ng1stloxPfragmentto50ulMLCK-PL253-EL350,pipettethemtoanelectroporationcup(0.1cMgap).
9.1.75kV,25uF,200ohms.
10.Add1mlSOCtoeachcurvetteandincubateat32℃for1h.
11.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernatantandplatecells(Amp+andK+).
$Kanamycinconcentration?
5.TransformMLCK1stloxPtoEL350togetpurifiedMLCK1stloxP(Amp+andK+)$?
?
Note:
EL350containsmultiplecopiesofplasmids,sotherewillbebothrecombinantandunrecombinantplasmidsinEL350.
6.1stloxPpopout(Amp+andK+Amp+)
7.PickupasinglecolonyofEL350to3mlLB,growat32℃O/N(12-16h)
8.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2htoOD600≈0.4.
9.InduceEL350with1mg/mlArabinoseat32℃for1h.
Fromthisstep,alloniceorin4℃
10.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min
11.Spinat3500rpmfor6min,resuspendcellswith1.5mlpH7.4MilliQ
12.Spin,washtwicemore
13.Removesupernatant,add50μlpH7.4MilliQ
8.AddMLCK1stloxPplasmid(dilution≈1:
1000)to50μlcompetentcells,pipettethemtoanelectroporationcup(0.1cMgap).
9.1.75kV,25μF,200ohms.
10.Add1mlSOCtoeachcurvetteandincubateat32℃for1h.
11.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernatant,platecells(Amp+).
Note:
YoucanplatecellstotheK+platetoconformthepopoutefficiency(NoorlittleelectroporatedcellsgrowontheK+plate)
7.TransformMLCK1stloxPpopouttoEL250(Amp+)
Note:
TheremaybeleakyexpressionoftheCrerecombinaseinEL350,leadingtoundesiredexcisionbetweenfloxedloxPsitesinthefinalconstruct.
8.Targeting2ndloxP(Amp+Amp+andK+)
Mini-targetingvectorconstruction
1.PCRamplify80bphomologousarmsFRT-Neo-FRT-loxPcassette(2ndloxP)fromPL451.
2.Ligate2ndloxPwithTvector(IfthePCRproductquantityisenoughforlateruse,thisprocedureisnotnecessary)
3.Cut1stloxP–Ttoget1stloxPfragment,purifytheproduct.
Note:
Ifyouusetheligationmethodtoconstructthemini-targetingvector,two~500bphomologousarmsareligatedtotheFRT-Neo-FRT-loxPcassetteexcisedfromPL451withEcoRIandBamHIandtoPL253thatislinearizedbyNotIandSalI.
Transformation
1.PickupasinglecolonyofMLCK1stloxPpopoutEL250to3mlLB,growat32℃O/N(12-16h)
2.Nextday,incubate1mlofO/Ncultureto50mlLB,32℃for2-3htoOD600=0.5
3.InduceMLCK1stloxPpopoutEL250in42℃waterbathbyshakingfor15min
Fromthisstep,alloniceorin4℃
4.Transfer6mlcellsto15mlcentrifugetube,putonicefor15min
5.Spinat3,500rpm6min,resuspendcellwith1.5mlpH7.4MilliQ
6.Spin,washtwicemore.
7.Removesupernatant,add50μlpH7.4MilliQ
8.Add200ng2ndloxpfragmentto50μlMLCK1stloxPpopoutEL250,pipettethemtoanelectroporationcup(0.1cMgap).
9.1.75kV,25μF,200ohms.
10.Add1mlSOCtoeachcurvetteandincubateat32℃for1h..
11.Centrifugeat3,500rpmfor3min,pouroffmostofthesupernatantandplatecells(Amp+andK+)
.
\
9.TransformMLCK2ndloxPtoDH-5αorXL1-Blue(Amp+andK+)topurifythetargetingvector
Itisimportanttoconfirmtherestrictionenzymepattern.
10.Linearization
SouthernBlotProtocol
1.Followingelectrophoresis,cuttheagarosegeltosizebyremovinganyblankoruntreatedlanesthatdonotneedtobetransferred.
2.Soakthegelinbasesolutionfor2×20minutes.Thereisnoneedtobetreatedwithneutralizingsolution.
3.AllowgenomicDNAtotransferovernightinbasesolution.
4.UVcrosslink,125mJ/cm2.Thenbakeat80℃for30minutes.
5.Hybridizethemembraneswithalabeledprobe.
Radioactivelabelingofprobe
(RediprimeIIRandomPrimeLabellingSystem,AmershamBiosciences)
1.DilutetheDNAtobelabeledtoaconcentrationof25ngin45ulofTEbuffer.
2.DenaturetheDNAsamplebyheatingto100℃for5minutesinaboilingwaterbath.
3.SnapcooltheDNAbyplacingonicefor5minutesafterdenaturation.
4.AddthedenaturedDNAtothereactiontube.
5.Add5ulofRedivue[32P]dCTPandmixbypepettingupanddownabout12times,movingthepipettetiparoundinthesolution.
6.Incubateat37℃for1hour,thenroomtemperatureovernight.
7.Add125ulEtOHand5ulNaAc,centrifugeat10000rpmfor10minutestoprecipitateDNA.DissolveDNAin50uldH2O.
8.Foruseinhybridization,denaturethelabeledDNAbyheatingto100℃for5minutes,thensnapcoolonicefor5minutes.(withoutsnapcooling,addhotprobeintohybridizationbuffer,itworksokey.)
Hybridization
Insertmembraneinhybridizationtubeandwetwith65℃hybridizationbuffer.Increasevolumeslightlytoadequatelycoverthemembrane(approximately5ml).Prehybrid
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