pLKO1PURO 慢病毒报装Protocols.docx
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pLKO1PURO 慢病毒报装Protocols.docx
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pLKO1PURO慢病毒报装Protocols
Protocols >pLKO.1Protocol
Addgene isaglobal,non-profitplasmidrepositorydedicatedtomakingiteasierforscientiststoshare.
pLKO.1– TRC CloningVector
AddgenePlasmid10878.ProtocolVersion1.0.December2006.
CopyrightAddgene2006,AllRightsReserved.Thisprotocolisprovidedforyourconvenience.See“warrantyinformation”inappendix.
TableofContents
∙A.pLKO.1-TRC CloningVector
∙A.1TheRNAiConsortium
∙A.2MapofpLKO.1
∙A.3Relatedplasmids
∙B.DesigningshRNAOligosforpLKO.1
∙B.1Determinetheoptimal21-mertargetsinyourgene
∙B.2OrderoligoscompatiblewithpLKO.1
∙C.CloningshRNAoligosintopLKO.1
∙C.1Recommendedmaterials
∙C.2Annealingoligos
∙C.3DigestingpLKO.1TRC-CloningVector
∙C.4Ligatingandtransformingintobacteria
∙D.ScreeningforInserts
∙D.1Recommendedmaterials
∙D.2Screeningforinserts
∙E.ProducingLentiviralParticles
∙E.1Recommendedmaterials
∙E.2Protocolforproducinglentiviralparticles
∙F.InfectingTargetCells
∙F.1Recommendedmaterials
∙F.2Determiningtheoptimalpuromycinconcentration
∙F.3Protocolforlentiviralinfectionandselection
∙G.Safety
∙H.References
∙H.1Publishedarticles
∙H.2Webresources
∙I.Appendix
∙I.1SequenceofpLKO.1TRC-CloningVector
∙I.2Recipes
∙I.3Warrantyinformation
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A.pLKO.1-TRC CloningVector
A.1TheRNAiConsortium
ThepLKO.1cloningvectoristhebackboneuponwhich TheRNAiConsortium hasbuiltalibraryofshRNAsdirectedagainst15,000humanand15,000mousegenes.Addgeneisworkingwiththe TRC tomakethisshRNAcloningvectoravailabletothescientificcommunity.PleaseciteMoffatetal.,Cell2006Mar;124(6):
1283-98('PubMed”:
http:
//www.ncbi.nlm.nih.gov/pubmed/16564017?
dopt=abstract)inallpublicationsarisingfromtheuseofthisvector.
A.2MapofpLKO.1
pLKO.1isareplication-incompetentlentiviralvectorchosenbythe TRC forexpressionofshRNAs.pLKO.1canbeintroducedintocellsviadirecttransfection,orcanbeconvertedintolentiviralparticlesforsubsequentinfectionofatargetcellline.Onceintroduced,thepuromycinresistancemarkerencodedinpLKO.1allowsforconvenientstableselection.
Figure1:
MapofpLKO.1containinganshRNAinsert.TheoriginalpLKO.1-TRC cloningvectorhasa1.9kbstufferthatisreleasedbydigestionwithAgeIandEcoRI.shRNAoligosareclonedintotheAgeIandEcoRIsitesinplaceofthestuffer.TheAgeIsiteisdestroyedinmostcases(dependingonthetargetsequence),whiletheEcoRIsiteispreserved.ForacompletemapofpLKO.1containingthe1.9kbstuffer,visit www.addgene.org/10878.
Description
VectorElement
U6
HumanU6promoterdrives RNA Polymerase III transcriptionforgenerationofshRNAtranscripts.
cPPT
Centralpolypurinetract,cPPT,improvestransductionefficiencybyfacilitatingnuclearimportofthevector’spreintegrationcomplexinthetransducedcells.
hPGK
Humanphosphoglyceratekinasepromoterdrivesexpressionofpuromycin.
PuroR
PuromycinresistancegeneforselectionofpLKO.1plasmidinmammaliancells.
sin3’LTR
3’Self-inactivatinglongterminalrepeat.
f1ori
f1bacterialoriginofreplication.
AmpR
AmpicillinresistancegeneforselectionofpLKO.1plasmidinbacterialcells
pUCori
pUCbacterialoriginofreplication.
5’LTR
5’longterminalrepeat.
RRE
Revresponseelement.
A.3RelatedProducts
ThefollowingplasmidsavailablefromAddgenearerecommendedforuseinconjunctionwiththepLKO.1TRC-cloningvector.
Plasmid(AddgeneID#)
Description
pLKO.1– TRC control
Negativecontrolvectorcontainingnon-hairpininsert.
pLKO.1–scrambleshRNA
NegativecontrolvectorcontainingscrambledshRNA.
psPAX2
Packagingplasmidforproducingviralparticles.
pMD2.G
Envelopeplasmidforproducingviralparticles.
Note:
pLKO.1canalsobeusedwithpackagingplasmid pCMV-dR8.2dvpr andenvelopeplasmid pCMV-VSVG fromRobertWeinberg’slab.Formoreinformation,visitAddgene’s MammalianRNAiTools page.
SeveralotherlaboratorieshavedepositedpLKOderivedvectorsthatmayalsobeusefulforyourexperiment.Toseethesevectors,visitAddgene’swebsiteand“searchfor“pLKO”“.
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B.DesigningshRNAOligosforpLKO.1
B.1DeterminingtheOptimal21-merTargetsinyourGene
Selectionofsuitable21-mertargetsinyourgeneisthefirststeptowardefficientgenesilencing.Methodsfortargetselectionarecontinuouslybeingimproved.Belowaresuggestionsfortargetselection.
1.UseansiRNAselectiontooltodetermineasetoftop-scoringtargetsforyourgene.Forexample,theWhiteheadInstituteforBiomedicalResearchhostsansiRNASelectionProgramthatcanbeaccessedafterafreeregistration(http:
//jura.wi.mit.edu/bioc/siRNAext/).IfyouhaveMacOSX,anotherexcellentprogramisiRNAi,whichisprovidedfreebythecompanyMekentosj(
AsummaryofguidelinesfordesigningsiRNAswitheffectivegenesilencingisincludedhere:
∙Startingat25ntdownstreamofthestartcodon(ATG),searchfor21ntsequencesthatmatchthepatternAA(N 19 ).Ifnosuitablematchisfound,searchforNAR(N 17 )YNN,whereNisanynucleotide,Risapurine(A,G),andYisapyrimidine(C,U).
∙G-C contentshouldbe36-52%.
∙Sense3’endshouldhavelowstability–atleastoneAorTbetweenposition15-19.
∙Avoidtargetingintrons.
∙Avoidstretchesof4ormorenucleotiderepeats,especiallyrepeatedTsbecausepolyTisaterminationsignalfor RNA polymerase III.
2.Tominimizedegradationofoff-targetmRNAs,useNCBI’s BLAST program.Selectsequencesthathaveatleast3nucleotidemismatchestoallunrelatedgenes.
Addgenerecommendsthatyouselectmultipletargetsequencesforeachgene.Somesequenceswillbemoreeffectivethanothers.Inaddition,demonstratingthattwodifferentshRNAsthattargetthesamegenecanproducethesamephenotypewillalleviateconcernsaboutoff-targeteffects.
B.2OrderingOligosCompatiblewithpLKO.1
TogenerateoligosforcloningintopLKO.1,insertyoursenseandantisensesequencesfromstepB.1intotheoligosbelow.Donotchangetheends;thesebasesareimportantforcloningtheoligosintothepLKO.1TRC-cloningvector.
Forwardoligo:
5’CCGG—21bpsense—CTCGAG—21bpantisense—TTTTTG 3’
Reverseoligo:
5’AATTCAAAAA—21bpsense—CTCGAG—21bpantisense3’
Forexample,ifthetargetsequenceis(AA)TGCCTACGTTAAGCTATAC,theoligoswouldbe:
Forwardoligo:
5’ CCGG AATGCCTACGTTAAGCTATAC CTCGAG GTATAGCTTAACGTAGGCATT TTTTTG 3’
Reverseoligo:
5’ AATTCAAAAA AATGCCTACGTTAAGCTATAC CTCGAG GTATAGCTTAACGTAGGCATT 3’
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C.CloningOligosintopLKO.1
ThepLKO.1-TRC cloningvectorcontainsa1.9kbstufferthatisreleasedupondigestionwithEcoRIandAgeI.
TheoligosfromsectionBcontaintheshRNAsequenceflankedbysequencesthatarecompatiblewiththestickyendsofEcoRIandAgeI.ForwardandreverseoligosareannealedandligatedintothepLKO.1vector,producingafinalplasmidthatexpressestheshRNAofinterest.
C.1RecommendedMaterials
Material
Vendorandcatalog#
AgeI
NewEnglandBiolabs(NEB)#R0552S
EcoRI
NEB #R0101S
T4 DNA ligase
NEB #M0202S
NEB buffer2
NEB #B7002S
DH5alphacompetentcells
Invitrogen#18258-012
Qiaquickgelextractionkit
Qiagen#28704
Lowmeltingpointagarose
Sigma#A9414
LuriaBrothAgar(LBagar)
AmericanBioanalytical:
#AB01200-02000
Ampicillin
VWR:
#7177-48-2.Useat100μg/mL.
Carbenicillin
VWR:
#80030-956.Useat100μg/mL.
C.2AnnealingOligos
1.ResuspendoligosinddH2Otoaconcentrationof20μM,thenmix:
5μLForwardoligo
5μLReverseoligo
5μL10x NEB buffer2
35μLddH2O
2.Incubatefor4minutesat95°Cina PCR machineorinabeakerofboilingwater.
3.Ifusinga PCR machine,incubatethesampleat70°Cfor10minutesthenslowlycooltoroomtemperatureovertheperiodofseveralhours.Ifusingabeakerofwater,removethebeakerfromtheflame,andallowthewatertocooltoroomtemperature.Thiswilltakeafewhours,butitisimportantforthecoolingtooccurslowlyfortheoligostoanneal.
C.3DigestingpLKO.1 TRC CloningVector
1.DigestpLKO.1TRC-cloningvectorwithAgeI.Mix:
6μgpLKO.1TRC-cloningvector(maxipreporminiprep DNA)
5μL10x NEB buffer1
1μLAgeI
to50μLddH2O
>Incubateat37°Cfor2hours.
2.PurifywithQiaquickgelextractionkit.Elutein30μLofddH2O.
3.DigesteluatewithEcoRI.Mix:
30μLpLKO.1TRC-cloningvectordigestedwithAgeI
5μL10x NEB bufferforEcoRI
1μLEcoRI
14μLddH2O
>Incubateat37°Cfor2hours.
4.Rundigested DNA on0.8%lowmeltingpointagarosegeluntilyoucandistinctlysee2bands,one7kbandone1.9kb.Cutoutthe7kbbandandplaceinasterilemicrocentrifugetube.
Whenvisualizing DNA fragmentstobeusedforligation,useonlylong-wavelengthUVlight.ShortwavelengthUVlightwillincreasethechanceofdamagingthe DNA.
5.Purifythe DNA usingaQiaquickgelextractionkit.Elutein30μLofddH2O.
6.Measurethe DNA concentration.
C.4LigatingandTransformingintoBacteria
1.Useyourligationmethodofchoice.ForastandardT4ligation,mix:
2μLannealedoligofromstepC.2.
20ngdigestedpLKO.1TRC-cloningvectorfromstepC.3.(Ifyouwereunabletomeasurethe DNA concentration,use1μL)
2μL10x NEB T4 DNA ligasebuffer
1μLNEB T4 DNA ligase
to20μLddH2O
>Incubateat16°Cfor4-20hours.
2.Transform2μLofligationmixinto25μLcompetentDH5alphacells,followingmanufacturer’sprotocol.PlateonLBagarplatescontaining100μg/mLampicillinorcarbenicillin(anampicillinanalog).
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D.ScreeningforInserts
Youmayscreenforplasmidsthatweresuccessfullyligatedbyrestrictionenzymedigestion.However,onceyouhaveidentifiedthepositiveclones,itis
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