基因敲除详细步骤.docx

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