pLKO.1 TRC Cloning Vector

A.1TheRNAiConsortium

ThepLKO.1cloningvectoristhebackboneuponwhichTheRNAiConsortium(TRC)hasbuiltalibraryofshRNAsdirectedagainst15,000humanand15,000mousegenes.AddgeneisworkingwiththeTRCtomakethisshRNAcloningvectoravailabletothescientificcommunity.PleaseciteMoffatetal.,Cell2006Mar;124(6):1283-98(PubMed)inallpublicationsarisingfromtheuseofthisvector.

A.2MapofpLKO.1

pLKO.1isareplication-incompetentlentiviralvectorchosenbytheTRCforexpressionofshRNAs.pLKO.1canbeintroducedintocellsviadirecttransfection,orcanbeconvertedintolentiviralparticlesforsubsequentinfectionofatargetcellline.Onceintroduced,thepuromycinresistanceMarkerencodedinpLKO.1allowsforconvenientstableselection.

Figure1:MapofpLKO.1containinganshRNAinsert.TheoriginalpLKO.1-TRCcloningvectorhasa1.9kbstufferthatisreleasedbydigestionwithAgeIandEcoRI.shRNAoligosareclonedintotheAgeIandEcoRIsitesinplaceofthestuffer.TheAgeIsiteisdestroyedinmostcases(dependingonthetargetsequence),whiletheEcoRIsiteispreserved.ForacompletemapofpLKO.1containingthe1.9kbstuffer,visitwww.addgene.org/10878.

Description	VectorElement
U6	HumanU6promoterdrivesRNAPolymeraseIIItranscriptionforgenerationofshRNAtranscripts.
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.

Figure2:DetailofshRNAinsert.TheU6promoterdirectsRNAPolymeraseIIItranscriptionoftheshRNA.TheshRNAcontains21"sense"basesthatareidenticaltothetargetgene,aloopcontaininganXhoIrestrictionsite,and21"antisense"basesthatarecomplementarytothe"sense"bases.TheshRNAisfollowedbyapolyTterminationsequenceforRNAPolymeraseIII.

A.3RelatedProducts

ThefollowingplasmidsavailablefromAddgenearerecommendedforuseinconjunctionwiththepLKO.1TRC-cloningvector.

Plasmid(AddgeneID#)	Description
pLKO.1-TRCcontrol(10879)	Negativecontrolvectorcontainingnon-hairpininsert.
pLKO.1-scrambleshRNA(1864)	NegativecontrolvectorcontainingscrambledshRNA.
psPAX2(12260)	Packagingplasmidforproducingviralparticles.
pMD2.G(12259)	Envelopeplasmidforproducingviralparticles.

Note:pLKO.1canalsobeusedwithpackagingplasmidpCMV-dR8.2dvpr(Addgene#8455)andenvelopeplasmidpCMV-VSVG(Addgene#8454)fromRobertWeinberg""slab.Formoreinformation,visitAddgene""sMammalianRNAiToolspage.

SeveralotherlaboratorieshavedepositedpLKOderivedvectorsthatmayalsobeusefulforyourexperiment.Toseethesevectors,visitAddgene""swebsiteandsearchfor"pLKO".

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B.1DeterminingtheOptimal21-merTargetsinyourGene

Selectionofsuitable21-mertargetsinyourgeneisthefirststeptowardefficientgenesilencing.Methodsfortargetselectionarecontinuouslybeingimproved.Belowaresuggestionsfortargetselection.

1.UseansiRNAselectiontooltodetermineasetoftop-scoringtargetsforyourgene.Forexample,theWhiteheadInstituteforBiomedicalResearchhostsansiRNASelectionProgramthatcanbeaccessedafterafreeregistration(http://jura.wi.mit.edu/bioc/siRNAext/).IfyouhaveMacOSX,anotherexcellentprogramisiRNAi,whichisprovidedfreebythecompanyMekentosj(http://www.mekentosj.com/irnai/).

AsummaryofguidelinesfordesigningsiRNAswitheffectivegenesilencingisincludedhere:

• Startingat25ntdownstreamofthestartcodon(ATG),searchfor21ntsequencesthatmatchthepatternAA(N₁₉).Ifnosuitablematchisfound,searchforNAR(N₁₇)YNN,whereNisanynucleotide,Risapurine(A,G),andYisapyrimidine(C,U).
• G-Ccontentshouldbe36-52%.
• Sense3""endshouldhavelowstability–atleastoneAorTbetweenposition15-19.
• Avoidtargetingintrons.
• Avoidstretchesof4ormorenucleotiderepeats,especiallyrepeatedTsbecausepolyTisaterminationsignalforRNApolymeraseIII.

2.Tominimizedegradationofoff-targetmRNAs,useNCBI""sBLASTprogram.Selectsequencesthathaveatleast3nucleotidemismatchestoallunrelatedgenes.

Addgenerecommendsthatyouselectmultipletargetsequencesforeachgene.Somesequenceswillbemoreeffectivethanothers.Inaddition,demonstratingthattwodifferentshRNAsthattargetthesamegenecanproducethesamephenotypewillalleviateconcernsaboutoff-targeteffects.

B.2OrderingOligosCompatiblewithpLKO.1

TogenerateoligosforcloningintopLKO.1,insertyoursenseandantisensesequencesfromstepB.1intotheoligosbelow.Donotchangetheends;thesebasesareimportantforcloningtheoligosintothepLKO.1TRC-cloningvector.

Forwardoligo:

5""CCGG—21bpsense—CTCGAG—21bpantisense—TTTTTG3""

Reverseoligo:

5""AATTCAAAAA—21bpsense—CTCGAG—21bpantisense3""

Forexample,ifthetargetsequenceis(AA)TGCCTACGTTAAGCTATAC,theoligoswouldbe:

Forwardoligo:

5""CCGGAATGCCTACGTTAAGCTATACCTCGAGGTATAGCTTAACGTAGGCATTTTTTTG3""

Reverseoligo:

5""AATTCAAAAAAATGCCTACGTTAAGCTATACCTCGAGGTATAGCTTAACGTAGGCATT3""

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ThepLKO.1-TRCcloningvectorcontainsa1.9kbstufferthatisreleasedupondigestionwithEcoRIandAgeI.

TheoligosfromsectionBcontaintheshRNAsequenceflankedbysequencesthatarecompatiblewiththestickyendsofEcoRIandAgeI.ForwardandreverseoligosareannealedandligatedintothepLKO.1vector,producingafinalplasmidthatexpressestheshRNAofinterest.

C.1RecommendedMaterials

Material	Vendorandcatalog#
AgeI	NewEnglandBiolabs(NEB)#R0552S
EcoRI	NEB#R0101S
T4DNAligase	NEB#M0202S
NEBbuffer2	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:

5μL	Forwardoligo
5μL	Reverseoligo
5μL	10xNEBbuffer2
35μL	ddH2O

2.Incubatefor4minutesat95^oCinaPCRmachineorinabeakerofboilingwater.

3.IfusingaPCRmachine,incubatethesampleat70^oCfor10minutesthenslowlycooltoroomtemperatureovertheperiodofseveralhours.Ifusingabeakerofwater,removethebeakerfromtheflame,andallowthewatertocooltoroomtemperature.Thiswilltakeafewhours,butitisimportantforthecoolingtooccurslowlyfortheoligostoanneal.

C.3DigestingpLKO.1TRCCloningVector

1.DigestpLKO.1TRC-cloningvectorwithAgeI.Mix:

6μg	pLKO.1TRC-cloningvector(maxipreporminiprepDNA)
5μL	10xNEBbuffer1
1μL	AgeI
to50μL	ddH2O

>Incubateat37^oCfor2hours.

2.PurifywithQiaquickgelextractionkit.Elutein30μLofddH₂O.

3.DigesteluatewithEcoRI.Mix:

30μL	pLKO.1TRC-cloningvectordigestedwithAgeI
5μL	10xNEBbufferforEcoRI
1μL	EcoRI
14μL	ddH2O

>Incubateat37^oCfor2hours.

4.RundigestedDNAon0.8%lowmeltingpointagarosegeluntilyoucandistinctlysee2bands,one7kbandone1.9kb.Cutoutthe7kbbandandplaceinasterilemicrocentrifugetube.

WhenvisualizingDNAfragmentstobeusedforligation,useonlylong-wavelengthUVlight.ShortwavelengthUVlightwillincreasethechanceofdamagingtheDNA.

5.PurifytheDNAusingaQiaquickgelextractionkit.Elutein30μLofddH₂O.

6.MeasuretheDNAconcentration.

C.4LigatingandTransformingintoBacteria

1.Useyourligationmethodofchoice.ForastandardT4ligation,mix:

2μL	annealedoligofromstepC.2.
20ng	digestedpLKO.1TRC-cloningvectorfromstepC.3.(IfyouwereunabletomeasuretheDNAconcentration,use1μL)
2μL	10xNEBT4DNAligasebuffer
1μL	NEBT4DNAligase
to20μL	ddH2O

>Incubateat16^oCfor4-20hours.

2.Transform2μLofligationmixinto25μLcompetentDH5alphacells,followingmanufacturer""sprotocol.PlateonLBagarplatescontaining100μg/mLampicillinorcarbenicillin(anampicillinanalog).

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Youmayscreenforplasmidsthatweresuccessfullyligatedbyrestrictionenzymedigestion.However,onceyouhaveidentifiedthepositiveclones,itisimportanttoverifytheinsertbyconductingasequencingreaction.

D.1RecommendedMaterials

Material	Vendorandcatalog#
DNAMiniprepKit	Qiagen#27104
EcoRI	NEB#R0101S
NcoI	NEB#R0193S
Agarose	Sigma#A9539

D.2ScreeningforInserts

Day1:

1.Innoculate5coloniesfromeachligationintoLB+100μg/mLampicillinorcarbenicillin.

Day2:

2.SpindowntheculturesanduseaminiprepkittoobtainDNA.

3.ConductarestrictiondigestwithEcoRIandNcoI:

1μg	miniprepDNA
2μL	10xNEBbufferforEcoRI
0.8μL	EcoRI
0.8μL	NcoI
to20μL	ddH2O

>Incubateat37^oCfor1-2hours.

4.Runthedigestionproductsona1%agarosegel.Youshouldseetwofragments,a2kbfragmentanda5kbfragment.

5.SequencepositivecloneswithpLKO.1sequencingprimer(5""CAAGGCTGTTAGAGAGATAATTGGA3"").

YoumayneedtoadjustthesequencingconditionsiftheDNApolymerasehasdifficultyreadingthroughthesecondarystructureofthehairpinsequence.

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E.ProducingLentiviralParticles

Beforethisstep,youmustcontactyourinstitution""sBio-Safetyofficetoreceivepermissionandinstitution-specificinstructions.Youmustfollowsafetyproceduresandworkinanenvironment(e.g.BL2+)suitableforhandlingHIV-derivativeviruses.

Fortransientknockdownofproteinexpression,youmaytransfectplasmidDNAdirectlyintothetargetcells.TheshRNAwillbeexpressed,buttheDNAisunlikelytobeintegratedintothehostgenome.

Forstableloss-of-functionexperiments,Addgenerecommendsthatyougeneratelentiviralparticlesandinfectthetargetcells.AdditionofpuromycinwillallowyoutoselectforcellsthatstablyexpressyourshRNAofinterest.

E.1RecommendedMaterials

Material	Vendorandcatalog#
psPAX2	Addgene#12260
pMD2.G	Addgene#12259
HEK-293Tcells	GenHunter:#Q401
FuGENE®6TransfectionReagent	RocheAppliedBiosciences:#11814443001
OPTI-MEM®serum-freemedia	Invitrogen:#31985
Dulbecco""sModifiedEagleMedium(DMEM)	Invitrogen:#11995
FetalBovineSerum(FBS)	Invitrogen:#16000
Penicillin/Streptomycin	Invitrogen:#15140-122
Polypropylenetubes	VWR:#87003-290

Note:pLKO.1couldalsobepackagedusingpCMV-dR8.2dvprandpCMV-VSVGfromtheRobertWeinberglab.Formoreinformation,visitAddgene""sMammalianRNAiToolspage.

E.2ProtocolforProducingLentiviralParticles

Thisprotocolisfortransfectionina6cmplate.Theprotocolcanbescaledtoproducedifferentamountsofvirusasneeded.

Day1:

a.Foreachplasmidtobetransfected,plate7x10⁵HEK-293Tcellsin5mLofmediaina6cmtissuecultureplate.Incubatecellsat37^oC,5%CO₂overnight.
AlthoughcellsshouldregularlybepassagedinDMEM+10%FBSwithpenicillin/streptomycin,cellsshouldbeplatedatthisstepinDMEM+10%FBSwithoutantibiotics(nopenicillinorstreptomycin).

Day2:

b.Performthetransfectioninthelateafternoonbecausethetransfectionmixshouldonlybeincubatedwiththecellsfor12-15hours.

c.Inpolypropylenemicrofugetubes(doNOTusepolystyrenetubes),makeacocktailforeachtransfection:

1μg	pLKO.1shRNAplasmid
750ng	psPAX2packagingplasmid
250ng	pMD2.Genvelopeplasmid
to20μl	serum-freeOPTI-MEM

YoumaywanttovarytheratioofshRNAplasmid,packagingplasmid,andenvelopeplasmidtoobtaintheratiothatgivesyoutheoptimalviralproduction.

d.CreateamastermixofFuGENE®6transfectionreagentinserum-freeOPTI-MEM.CalculatetheamountofFugene®andOPTI-MEMnecessarygiventhateachreactionwillrequire6μLFuGENE®+74μLOPTI-MEM.Forexample:

1xmastermix:	6μLFuGENE®+74μLOPTI-MEM
5xmastermix:	30μLFuGENE®+370μLOPTI-MEM
10xmastermix:	60μLFuGENE®+740μLOPTI-MEM

Inapolypropylenetube,addOPTI-MEMfirst.PipetteFuGENE®directlyintotheOPTI-MEM-donotallowFuGENE®tocomeincontactwiththewallsofthetubebeforeithasbeendiluted.Mixbyswirlingorgentlyflickingthetube.Incubatefor5minutesatroomtemperature.

e.Add80μLofFuGENE®mastermixtoeachtubefromstepcforatotalvolumeof100μL.Pipettemastermixdirectlyintotheliquidandnotontothewallsofthetube.Mixbyswirlingorgentlyflickingthetube.

f.Incubatefor20-30minutesatroomtemperature.

g.RetrieveHEK-293Tcellsfromincubator.Thecellsshouldbe50-80%confluentandinDMEMthatdoesnotcontainantibiotics.

h.Withouttouchingthesidesofthedish,gentlyaddDNA:FuGENE®mixdropwisetocells.Swirltodispersemixtureevenly.Donotpipetteorswirltoovigorously,asyoudonotwanttodislodgethecellsfromtheplate.

i.Incubatecellsat37^oC,5%CO₂for12-15hours.

Day3:

j.Inthemorning,changethemediatoremovethetransfectionreagent.Replacewith5mLfreshDMEM+10%FBS+penicillin/streptomycin.Pipettethemediaontothesideoftheplatesoasnottodisturbthetransfectedcells.

k.Incubatecellsat37^oC,5%CO₂for24hours.

Day4:

l.Harvestmediafromcellsandtransfertoapolypropylenestoragetube.Themediacontainsyourlentiviralparticles.Storeat4^oC.

m.Add5mLoffreshmediacontainingantibioticstothecellsandincubateat37^oC,5%CO₂for24hours.

Day5:

n.HarvestmediafromcellsandpoolwithmediafromDay4.Spinmediaat1,250rpmfor5minutestopelletanyHEK-293Tcellsthatwereinadvertentlycollectedduringharvesting.
Inlieuofcentrifugation,youmayfilterthemediathrougha0.45μmfiltertoremovethecells.Donotusea0.2μmfilter,asthisislikelytosheartheenvelopeofyourvirus.

o.Virusmaybestoredat4^oCforafewdays,butshouldbefrozenat-20^oCor-80^oCforlong-termstorage.

Freeze/thawcyclesdecreasetheefficiencyofthevirus,soAddgenerecommendsthatyouusethevirusimmediatelyoraliquotthemediaintosmallertubestopreventmultiplefreeze/thawcycles.

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F.InfectingTargetCells

Lentiviralparticlescanefficientlyinfectabroadrangeofcelltypes,includingbothdividingandnon-dividingcells.AdditionofpuromycinwillallowyoutoselectforcellsthatarestablyexpressingyourshRNAofinterest.

F.1.RecommendedMaterials

Material	Vendorandcatalog#
Hexadimethrinebromide(polybrene)*	Sigma-Aldrich:#H9268
ProtamineSulfate*	MPBiomedicals:#194729
Puromycin*	Sigma-Aldrich:#P8833
Targetcells	Variesbasedonyourexperiment
Culturemediafortargetcells	Variesbasedonyourexperiment
Materialsforassay	Variesbasedonyourexperiment

*Detailedprotocolsforpreparingpolybrene,protaminesulfate,andpuromycinarelocatedintheAppendix.

F.2.DeterminingtheOptimalPuromycinConcentration

Eachcelllinerespondsdifferentlytopuromycinselection.Addgenestronglyrecommendsthatyoudeterminetheoptimalpuromycinconcentrationforyourcelllinebeforeinitiatingyourexperiment.

Day1:

a.Platetargetcellsinten6cmplatesandgrowat37^oC,5%CO₂overnight.

Day2:

b.Thetargetcellsshouldbeapproximately80-90%confluent.

c.Dilutepuromycininthepreferredculturemediaforyourtargetcells.Thefinalconcentrationofpuromycinshouldbefrom1-10μg/mLin1μg/mLincrements.

d.Labelplatesfrom1-10andaddappropriatepuromycin-containingmediatocells.

Days3+:

e.Examinecellseachdayandchangetofreshpuromycin-containingmediaeveryotherday.

f.Theminimumconcentrationofpuromycinthatresultsincompletecelldeathafter3-5daysistheconcentrationthatshouldbeusedforselectioninyourexperiments.(Youmaywishtorepeatthistitrationwithfinerincrementsofpuromycintodetermineamorepreciseoptimalpuromycinconcentration.)

F.3.ProtocolforLentiviralInfectionandSelection

Day1:

a.Platetargetcellsandincubateat37^oC,5%CO₂overnight.

Day2:

b.Targetcellsshouldbeapproximately70%confluent.Changetofreshculturemediacontaining8μg/mLpolybrene.
Polybreneincreasestheefficiencyofviralinfection.However,polybreneistoxictosomecelllines.Inthesecelllines,substituteprotaminesulfateforpolybrene.

c.AddlentiviralparticlesolutionfromstepE.Fora6cmtargetplate,addbetween0.05-1mLvirus(add≥0.5mLforahighMOI,and≤0.1mLforalowMOI).Scaletheamountofvirusaddeddependingonthesizeofyourtargetplate.

MOI(multiplicityofinfection)referstothenumberofinfectingviralparticlespercell.AddgenerecommendsthatyoutestarangeofMOIstodeterminetheoptimalMOIforinfectionandgenesilencinginyourtargetcellline.

d.Incubatecellsat37^oC,5%CO₂overnight.

Day3:

e.Changetofreshmedia24hoursafterinfection.
Ifviraltoxicityisobservedinyourcellline,youmaydecreasetheinfectiontimetobetween4-20hours.Removethevirus-containingmediaandreplacewithfreshmedia.Donotaddpuromycinuntilatleast24hoursafterinfectiontoallowforsufficientexpressionofthepuromycinresistancegene.

f.Toselectforinfectedcells,addpuromycintothemediaattheconcentrationdeterminedinstepE.2.

Addgenerecommendsthatyoumaintainoneuninfectedplateofcellsinparallel.Thisplatewillserveasapositivecontrolforthepuromycinselection.

Days4+:

g.Changetofreshpuromycin-containingmediaasneededeveryfewdays.

h.Assayinfectedcells.Thefollowingrecommendationsareguidelinesforthenumberofdaysyoushouldwaituntilharvestingyourcells.However,youshouldoptimizethetimebasedonyourcelllineandassay:

Assay	Dayspost-infection
mRNAknockdown(quantitativePCR)	≥3days
Proteinknockdown(westernblot)	≥4days
Phenotypicassay	≥4days

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G.Safety

BL2safetypracticesshouldbefollowedwhenpreparingandhandlinglentiviralparticles.Personalprotectiveclothingshouldbewornatalltimes.Useplasticpipettesinplaceofglasspipettesorneedles.Liquidwasteshouldbedecontaminatedwithatleast10%bleach.Laboratorymaterialsthatcomeincontactwithviralparticlesshouldbetreatedasbiohazardouswasteandautoclaved.PleasefollowallsafetyguidelinesfromyourinstitutionandfromtheCDCandNIHforworkinaBL2facility.

Ifyouhaveanyquestionsaboutwhatsafetypracticetofollow,pleasecontactyourinstitution""ssafetyoffice.

ToobtaintheMSDSforthisproduct,visitwww.addgene.org/sitemapandfollowtheMSDSlink.

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H.References

H.1.PublishedArticles

KhvorovaAet.al.2003.FunctionalsiRNAsandmiRNAsexhibitstrandbias.Cell115:209-216.(PubMed)

MoffatJet.al.2006.AlentiviralRNAilibraryforhumanandmousegenesappliedtoanarrayedviralhigh-contentscreen.Cell124:1283-1298.(PubMed)

NaldiniLet.al.1996.Invivogenedeliveryandstabletransductionofnondividingcellsbyalentiviralvector.Science272:263-267.(PubMed)

SchwarzDSet.al.2003.AsymmetryintheassemblyoftheRNAienzymecomplex.Cell115:199-208.(PubMed)

StewartSAet.al.2003.Lentivirus-deliveredstablegenesilencingbyRNAiinprimarycells.RNA9(4):493-501.(PubMed)

ZuffereyRet.al.1997.Multiplyattenuatedlentiviralvectorachievesefficientgenedeliveryinvivo.NatBiotechnol15(9):871-5.(PubMed)

ZuffereyRet.al.1998.Self-inactivatinglentivirusvectorforsafeandefficientinvivogenedelivery.JVirol72(12):9873-80.(PubMed)

H.2.Webresources

Addgene""smammalianRNAiwebsite:www.addgene.org/rnaitools

TheRNAiConsortium(TRC):www.broad.mit.edu/genome_bio/trc/rnai.html

BackgroundonRNAimechanism:www.nature.com/focus/rnai/animations/animation/animation.htm

WhiteheadsiRNASelectionProgram:jura.wi.mit.edu/bioc/siRNAext/

MekentosjiRNAiProgram:www.mekentosj.com/irnai/

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I.Appendix

I.1.SequenceofpLKO.1TRC-CloningVector

Clickhere(http://www.addgene.org/10878)toseethesequenceofpLKO.1TRC-cloningvector.Thevectoris8901basepairstotal,andthestufferinsertisshowninallcapitalletters.

I.2.Recipes

LuriaBrothAgar(LBagar)+antibiotic

Per40gramsofpowderfromAmericanBioanalyticalcatalog#AB01200-02000,LBcontains:

10g	tryptone
5g	yeastextract
10g	sodiumchloride
15g	agar

>PrepareLBagarsolutionbydissolving40gofLBpowderin1Lofdistilledwater.Autoclaveandcoolto55^oC.Add1mLof100mg/mLampicillinorcarbenicillintoobtainafinalconcentrationof100μg/mLantibiotic.Pourplatesandstoreat4^oC.

HexadimethrineBromide(Polybrene)

Preparea1mg/mLsolutionofpolybrene(Sigma-Aldrichcatalog#H9268)in0.9%NaCl.Autoclavetosterilize.Stocksolutionisstableat4^oCforuptooneyear.Thepowderformofpolybreneisstableat4^oCforseveralyears.

ProtamineSulfate

Storeprotaminesulfate(MPBiomedicalscatalog#194729)at4^oC.Freelysolubleinhotwaterandslightlysolubleincoldwater.

Puromycin

Preparea50mg/mLstocksolutionofpuromycin(Sigma-Aldrichcatalog#P8833)indistilledwater.Sterilizebypassingthrougha0.22μmfilter.Storealiquotsat-20^oC.

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