InFusionCloningTips

InFusionCloningisahighlyefficient,ligationindependentmethodbasedontheannealingofcomplementaryends
ofacloninginsertandlinearizedcloningvector.Thistechnologyensureseasy,singlestepdirectionalcloningofany
geneofinterestintoanyvectoratanylocus.InFusionconstructsareseamless,enablingtranslationalreadingframe
continuitywithoutanyinterferingscarsequences.
ThefollowinginformationappliestocurrentInFusionCloningkits:InFusionHDCloningPlus,InFusionHD
CloningPlusCE,andInFusionHDEcoDryCloningPlus.

GENERAL
INFO

VECTORS/
INSERTS/
PRIMERS

MISCOPTIONS/
PCR/
SELECTED
GUIDELINES
TRANSFORMATION APPLICATIONS

GeneralInformation
Planningyourexperiment
SuccessfulInFusionCloningreactionsrequire15bphomologousoverlapsattheterminiofthecloninginsert
andlinearizedvector,oradjacentinsertsifmultipleinsertsaretobejoinedsimultaneously.
TheseoverlapscanbegeneratedbyPCRamplificationoroligosynthesisofeitherofthecloningfragments.
Homologousoverlapsshorterthan12ntorlongerthan21ntarenotrecommended.
Translationalreadingframecontinuityofafusionconstructcanbeadjustedbyaddingnucleotidesbetweenthe
insertspecificsequenceand15ntoverlap.
15bpcomplementaryregionsmustbelocatedattheterminiofadjacentDNAfragmentsortheywillnotbejoined
byInFusionCloning.

A3'exonucleaseintheInFusionenzymemixgenerates15ntsinglestranded5'overhangsattheterminiofthe
cloninginsertandlinearizedvector.Theseoverhangsareannealedatthesitesofcomplementarity,andthe
recombinantcircularconstructisrescuedinE.coli.
Wedonotrecommenduseofcellswithcompetencylessthan108 cfu/gsupercoiledDNA.
InFusionCloningdoesnotallowforthecovalentassemblyoflinearDNAmolecules.

Choosingakitformat
InFusionHDCloningPlusKitsareavailableinalyophilized(EcoDry)orliquidformat.Eachofthesekits
containCloneAmpHiFiPCRPremix,InFusionHDenzymepremix,andreagentsforcontrolexperiments
(linearizedvector,2kbinsert).
Therearetwodecisionstomakewhendecidingonakitformat.First,doyouwantalyophilizedorliquidkitformat
andsecond,whatPCRproductpurificationmethodissuitableforyouramplifiedDNAinsert?Thetablebelow
illustratesthedifferencesbetweenthelyophilizedandliquidkitformats.Afteryoumakethisdecision,continue
readingtolearnthedifferentoptionsforPCRproductpurification.

Lyophilizedversusliquidkitformats
Feature

InFusionHDEcoDryCloningPlus

InFusionHDCloningPlus

Prealiquoted,lyophilizedcomponents
thatminimizehandlingerrors
Roomtemperaturestorage,saving
freezerspaceandeliminatingfreeze
thawcycles
Abilitytocustomize:reactionvolumes
and/orplasticware
30minutereactiontime
15minutereactiontime

PurificationofPCRproducts:
ItisimportanttopurifyPCRproductsandvectorspriortocombininginthecloningreaction.BothInFusionHD
EcoDryCloningPlusandInFusionHDCloningPluskitsincludeaNucleoSpinGelandPCRCleanUpkitfor
purificationofPCRproducts.ThesekitsaresuitableforPCRandgelextractioninsituationswherePCRproduces
morethanoneproduct.
InFusionHDCloningPlusCEcontainsallthekitcomponentsprovidedinotherInFusionCloningPluskits,
butCloningEnhancer(CE)isincludedfortreatmentofPCRproductsinplaceofNucleoSpinGelandPCRClean
Up.CEisaproprietaryenzymemixforremovingbackgroundplasmidDNAandPCRresidue,thuseliminatingthe
needforadditionalpurificationofPCRamplifiedDNApriortotheInFusionreaction.UseofCEisonly
appropriateifPCRamplificationgeneratesasinglePCRfragmentoftheexpectedsize,withoutabackground
smear.CEisaconvenienttoolforhighthroughput(HTP)applicationsthatemployhighlyoptimizedPCRcycling
conditionsandprimersthatgeneratecleanproductsoftheexpectedsize.

Vectors
Compatiblevectors
AnylinearvectorcanbeusedforInFusionCloning,regardlessofsize,composition,oravailablerestriction
site(s).ThecloningreactionisfollowedbytherescueofacircularrecombinantconstructinE.coli,butplease
notethatcircularvectorsarenotcompatiblewiththecloningreactionitself.Additionally,InFusionCloningdoes
notallowtheassemblyofcovalentlylinkedlinearDNAmolecules.

Vectorsize
InFusiontechnologyallowseasycloningofsingleormultipleDNAfragmentsdirectlyintolargevectors(e.g.,
adenoviralvectorsat32.636kb,cosmidsat46kb*)inasinglereaction,withoutintermediatecloninginto
transfer/shuttlevectors.(PleaseseeFigures1,2,5,andTableIIIoftheAdenoXAdenoviralSystem3
Brochurefordetails.)
* The46kbcosmidvector,assembledbyInFusionHDCloning,wastransformedinchemicallycompetentbacteria,allowingthe

rescueoftherecombinantvector.Itwasnotusedintheactualcosmidpackagingreaction.

Vectorlinearizationandpurification
Linearizationoptionsinclude:
Restrictiondigestwithoneormorerestrictionenzymes.
ForefficientInFusionCloning,integrityofthelinearizedvectorterminiisessential.Werecommendusinghigh
qualityrestrictionenzymes,andperformingdigestsoverseveralhours.However,overnightrestrictiondigestis
notadvisable.
DephosphorylationofthevectorterminiisnotrequiredthevectorwillnotrecircularizeintheInFusionCloning
reactionmixunlessitcarries15ntcomplementaryoverlapsatitstermini.
Vectorslinearizedviarestrictiondigestshouldbepurifiedbypreparativeagarosegelelectrophoresis(covered
withaluminumfoiltopreventDNAdamage).Electrophoresisshouldbeperformedatalowvoltagetoensurethe
separationoflinearandcircular(uncut)vectormolecules.
InversePCRwithprimerspositionedatthedesiredcloningsite.
Choiceofcloninglocusisflexible,sincesuitablerestrictionsitesarenotrequired.
SimultaneousPCRmediatedmutagenesis(deletion,insertion,basechange)ispossible.(Pleasesee
ourprerecordedwebinaronthisapplication.)
The15bphomologousoverlapscanbeaddedtothePCRlinearizedvectorinsteadoftheinsert.
PreservetheintegrityofthevectorbackbonebyusingaPCRpolymerasewithhighproofreadingactivity,
likeCloneAmpHiFiPCRPremix(suppliedwithInFusionHDCloningPluskits).Thispolymeraseishighly
robustandaccurate,enablingamplificationofupto6kbofhumangenomicDNA,10kbofE.coligenomicDNA,
and15kbofLambdaDNA.ItiscompatiblewithtwoorthreestepPCRcycling,andexhibitsminimalerrorrates
onGCrichtemplates.

MutationfrequencyofCloneAmpHiFiPolymerasecomparedtootherhighfidelityPCRenzymes.Eight
arbitrarilyselectedGCrichregionswereamplifiedwithCloneAmpHiFiPolymeraseorotherDNApolymerases
usingaThermusthermophilusHB8genomicDNAtemplate,andclonedintosuitableplasmids.Multipleclones
wereselectedforeachamplificationproductandsubjectedtosequenceanalysis.DNAfragmentsamplified
usingCloneAmpHiFiPolymeraseyieldedonly12mismatchedbasesper542,580totalbaseslowerthanan
alternativehighfidelityenzymefromCompanyA,and10foldlowerthanTaqDNApolymerase.

VectorslinearizedviainversePCRshouldbetreatedwithCloningEnhancer(CE)todestroytheparental
plasmid.CEtreated,PCRlinearizedvectorsmayrequireadditionalpurificationbyagarosegelelectrophoresis
ifPCRbyproductsarepresentinthelinearizedvectorprep.

Inserts
Insertsources
ThefollowingtypesofinsertsarecompatiblewithInFusionCloning:
PCRamplifiedDNAfragmentscarryingoverhangscomplementarywiththeterminioftheadjacentDNA
fragment(s),syntheticoligos,orlinearvector.
DNAfragmentsgeneratedbyrestrictiondigestwithoneormorerestrictionenzymes.Inthisinstance,therequired
15bphomologymustbecarriedbytheadjacentDNAfragment(s),syntheticoligos,orlinearvector.
Syntheticoligonucleotides(50bp)with15bphomologytotheterminiofadjacentfragmentsorthelinearized
vector.Highquality,nonphosphorylatedoligonucleotidespurifiedbydesaltingarecompatiblewithInFusion
Cloning.GelorHPLCpurificationofoligonucleotidesisnotrequired.

Largeinserts
InFusiontechnologyhasbeenoptimizedforcloninglargefragments.DNAinsertsupto15kbhavebeen
successfullyclonedintopUC19usingInFusionCloning.

Tenoutoftencoloniescontainthecorrectinsert(100%efficiency)whencloningfragmentsaslargeas15kb
(resultsconfirmedbycolonyPCRscreening).

Smallinserts
ThesmallestinsertsuccessfullyclonedwithInFusionCloningwasa50bpsyntheticoligonucleotide(including
two15nthomologousoverlapswiththevectortermini).
ForInFusionCloningofshortsyntheticoligos(between50and150bp),thesuggestedoligotovectormolarratio
is515:1.Dependingonoligolength,theoptimalratiomustbedeterminedempirically.
Note:NonphosphorylatedoligonucleotidesarecompatiblewithInFusionCloning.However,the3'exonuclease
activityintheInFusionenzymemixrequiresterminal3'OHgroups.

Multipleinserts
Wehavesuccessfullytestedmultiplefragmentcloningwithuptofiveinserts.(SeeFigureandTablebelowfor
cloningschematicandcolonyscreenresults,respectively.)

PrimerdesignformultiplefragmentcloningcanbedonewithouronlinePrimerDesignTool.(ThePrimerDesign
TooliscompatiblewithMozillaFirefoxorGoogleChromewebbrowsers,butnotwithInternetExplorer.)Wealso
haveaPrimerDesignTooltutorialspecificallyformultiplefragmentcloning.
Pleasenotethatbetweentwoadjacentfragments,onlyonehomologousoverlapisrequiredfortheInFusion
reaction.Thisoverlapcanbelocatedoneitherofthefragments.

TheInFusionHDCloningSystemhasanimprovedcapabilityforcloningmultiplefragmentsinasingle
reaction.Usingthissystem,cloninguptofour1kbfragmentssimultaneouslyisaseasyascloningasinglefragment.
Thissavesweeksthatwouldotherwisebespentscreeningclonesandsubcloning.

Insert

ColonyScreening

Fragments

Colonies,1/5plated

Correctclones

1kb+1kb

2,128

10/10

1kb+1kb+1kb

83

7/10

1kb+1kb+1kb+1kb

31

8/10

1kb+1kb+1kb+1kb+1kb

14

4/10

PrimerDesign
PCRprimerscompatiblewithInFusionCloning
Eachforward(5'3'sensestrand)andreverse(5'3'antisensestrand)InFusionCloningPCRprimershould
includethefollowing:
Templatespecific(genespecific)portionatits3'end.ToensurespecificandefficientPCRamplification,the
templatespecificportionoftheprimershouldbe1825ntinlength.
15ntofhomologyatthe5'endoftheprimer,complementarytotheterminiofthelinearizedvectororadjacent
inserts(ifmultipleinsertsaretobeclonedsimultaneously).Homologousoverlapsshorterthan12ntandlonger
than21ntarenotrecommended.The15bpcomplementaryregionsmustbelocatedattheterminiofadjacent
DNAfragmentsortheywillnotbejoinedbyInFusionCloning.
Note:Whenavectorhasbeenlinearizedviarestrictiondigest,the5'overhangofarestrictionsiteisincludedin
the15ntofhomology,whilethe3'overhangofarestrictionsiteisexcludedfromthecountofthe15ntof

homology.
(Optional)Toensurecontinuityofthetranslationalreadingframe,ortopreserverestrictionsite(s),additional
nucleotidescanbeaddedtothePCRprimer(s)betweenthetemplatespecificportionandthe15nthomologous
overlap.

GeneratinghomologousoverlapsofDNAfragments
15bphomologousoverlapsbetweencloningterminifacilitateallInFusionCloningreactions.Theycanbe
generatedinthefollowingways:
PCRamplificationofacloninginsertusingPCRprimerscarrying15nt5'overhangsthatarehomologoustothe
terminiofthelinearizedvectororadjacentinsert
PCRlinearizationofthedestinationvector,usingPCRprimerscarrying15nt5'overhangshomologouswiththe
cloninginsert(s)
Oligonucleotidesynthesis,generating15nt5'endoverhangshomologouswiththeterminiofthelinearizedvector
oradjacentinsert.Highquality,nonphosphorylatedoligonucleotidespurifiedbydesaltingarecompatiblewith
InFusionCloning.GelorHPLCpurificationofoligonucleotidesisnotrequired.

Primerdesigntools
InstructionsfordesigningInFusionPCRprimersareincludedinallInFusionCloningusermanuals.Several
toolsarealsoavailabletohelpwiththeprocess.
OuronlinePrimerDesignToolfacilitatesprimerdesignforInFusionCloning,andiscompatiblewithMozilla
FirefoxorGoogleChromewebbrowsers.(InternetExplorerisnotcompatiblewiththePrimerDesignTool.)Specific
cloningconditionssupportedareasfollows:
Singlefragmentcloning
Multiplefragmentcloning
Vectorslinearizedbyrestrictiondigest
VectorslinearizedbyinversePCR
Prelinearizedvectors
Insertspecificprimersdesignedbytheuser
InsertspecificprimersgeneratedbythePrimerDesignTool
StepbysteptutorialsforthePrimerDesignToolarealsoavailableintheCloningResourcessectionofour
website.
ThecurrentversionofthePrimerDesignTooldoesnotallowadjustmentfortranslationalreadingframecontinuity,
whichshouldbemanuallydesignedbytheuser.
WealsorecommendSnapGeneViewerasahelpful,freeonlinetoolforinsilicoassemblyofyourrecombinant
construct,manualdesignofInFusionPCRprimers,andadjustmentoftranslationalreadingframecontinuity.

MiscellaneousOptions
Restrictionsitepreservation
Primerdesignletsyoueasilypreserveoreliminatetherestrictionsitesusedtolinearizethecloningvector.In
ordertomaintainrestrictionsitesatcloningjunctions,nucleotidescanbeaddedtothePCRprimersbetweenthe
templatespecificportionandthe15nthomologousoverlap.
TheonlinePrimerDesignToolofferstheoptiontopreservetherestrictionsite(s).(ThePrimerDesignToolis
compatiblewithMozillaFirefoxandGoogleChromewebbrowsers,butnotwithInternetExplorer.)

Translationalreadingframes
InFusionCloningmakesitpossibletoseamlesslycloneyourgeneofinterestinthesametranslationalreading
frameasadesiredtag(e.g.,fluorescentprotein,Myc,HA,etc.).SpecificsinthedesignofInFusionPCRprimers
facilitatethisapplication,allowingyoutomaintainreadingframecontinuityintherecombinantvector.Twooptions
fordoingsoarebelow:
Adjustingthelengthofthetemplatespecific(genespecific)portionoftheInFusionPCRprimer
Addingnucleotidesbetweenthetemplatespecificportionandthe15nthomologousoverlapportionofthe
InFusionPCRprimer

PleasenotethatthecurrentversionofouronlinePrimerDesignTooldoesnotallowadjustmentfortranslational
readingframecontinuity.Theprimersequenceshouldbemanuallydesignedbytheuser.We
recommendSnapGeneViewerasahelpful,freeonlinetooltohelpwiththistask.

Insertingexternalnucleotidesequences
Smallexternalnucleotidesequences(e.g.,smalltags,Kozaksequences,restrictionsites,cleavagesites,etc.)
canbeaddedbetweenthetemplate/genespecificportionandthe15nthomologousoverlapoftheInFusion
PCRprimer.
OurInFusionWebinarSeriesincludesaprerecordedvideospecifictothisapplication.

Splittingthe15nthomologousoverlap
Thehomologous15ntoverlapcanbesplitbetweentwoadjacentDNAfragments.Itcanonlybesplitbetweenan
insertandvectorifthevectorislinearizedviainversePCR.
PrimerdesignforthisoptionisnotfacilitatedbytheonlinePrimerDesignTool.Werecommend SnapGene
Viewerasahelpful,freeonlinetooltohelpwiththistask.

ThediagrambelowshowsInFusionprimerdesignandtheannealingofcomplementarystrands,usinga15nt
overlapsplitbetweenFragment1(red)andFragment2(blue):

Sitedirectedmutagenesis
InFusionCloningallowssingleormultiplebasechanges,deletions,andinsertions.Fordetails,pleasesee
ourprerecordedmutagenesiswebinarand/ortheMutagenesiswithInFusionHDCloningPlustechnote.

GeneralGuidelines
Molarratios
InFusionHDCloningPlususesaveryrobustenzyme,andallowshighlyefficientcloninginmostsituations.
Generalrecommendationsoninsert/vectorquantitiesareincludedinallcurrentInFusionCloningusermanuals.
Toensureoptimalresultsunderstandardconditions,orwhenperformingsingleormultiplefragmentcloning,use
aninserttovectorratioof2:1.
Themolarratioofeachofthemultipleinsertsshouldbe2:1withregardstothelinearized,purifiedvector.The
molarratiooftwoinsertswithonevectorshouldbe2:2:1.
TocalculatetherequiredamountofeachoftheDNAfragments,usenolessthan20ngofthesmallestinsert
andcalculatethequantitiesoftherestofthefragmentsaccordingly,maintainingthe2:1inserttovectormolar
ratio.(Eachoftheinsertsshouldbecalculatedatthe2:1molarratiowithregardtothevector.)
ForcloningofsmallDNAfragments(between150and350bp),thesuggestedinserttovectormolarratiois35:1.
Forcloningofshortsyntheticoligos(between50bpand150bp),thesuggestedoligotovectormolarratiois5
15:1.Dependingontheoligolength,theoptimalmolarratiomustbedeterminedempirically.
NonphosphorylatedoligonucleotidesarecompatiblewithInFusionCloning.However,3'exonucleaseactivity
intheInFusionenzymemixrequiresterminal3'OHgroups.
UseouronlineMolarRatioCalculatortocalculatespecificinserttovectorquantitiesbasedonmolarratios,insert
length(bp),andvectorlength(bp).

Controlreactions
AlwaysperformacontrolInFusionCloningreactionusingthecontrolvector(linearizedpUC19)andcontrolinsert
providedineachkit.Ifyourexperimentproducesunexpectedresults,thecontrolreactioncanhelpyouto
determinewheretostarttroubleshooting.
Thenegativecontrolprovidedwiththekittypicallyproducesfewerthan5%bluecoloniesthenumberofwhite
coloniesproducedvariesslightlydependingonthebacterialstrainusedfortransformation.Ingeneral,fewerthan
5%ofthewhitecoloniesonanexperimentalplatecontainbackground.Ithasbeenourobservationthat95%of
thecoloniesonexperimentalplatesarecorrect.ThisspeakstoInFusiontechnology'shighlevelofcloning
efficiency,i.e.,thepercentageofcorrectcoloniesrecoveredregardlessofthetotalnumberoftransformed
coloniespresent.

Incubationtime
AnincreaseintheInFusionreactiontimeisnotrecommended.Itmaygenerateunevensinglestrandedregions
attheendsofthecloninginsertandvector,resultingininefficientannealingofthehomologousoverlaps,thus
reducingcloningefficiency.

Locationofhomologousoverlaps
Thehomologous15bpoverlapsshouldbelocatedpreciselyattheterminiofthevectorandinsert.15bp
complementaryregionsnotlocatedattheterminiofadjacentDNAfragmentswillnotbejoinedbyInFusion
Cloning.PCRlinearizationofavectorallowspositioningoftheprimersatthedesiredcloninglocus,regardlessof
availablerestrictionsites,thusenablingthegenerationofthe15bpoverlapsattheterminiataparticularposition.

Lengthofhomologousoverlaps
CurrentInFusionCloningreactionconditionsfavora15bphomologousoverlap.Wedonotrecommendusing
overlapsshorterthan12bporlongerthan21bp.

PCRRequirements
Compatiblepolymerases
InFusionCloningiscompatiblewithanyPCRpolymerase.3'overhangsdonotinterferewiththecloning
reaction.
Toensureanerrorfreeinsert,useapolymerasewithhighproofreadingactivity,likeCloneAmpHiFiPCR
Premix(suppliedwithallcurrentInFusionCloningkits).Thispolymeraseishighlyrobustandaccurate,enabling
amplificationofupto6kbofhumangenomicDNA,10kbofE.coligenomicDNA,and15kbofLambdaDNA.Itis
compatiblewithtwoorthreestepPCRcycling,andexhibitsminimalerrorratesonGCrichtemplates.

MutationfrequencyofCloneAmpHiFiPolymerasecomparedtootherhighfidelityPCRenzymes.Eightarbitrarily
selectedGCrichregionswereamplifiedwithCloneAmpHiFiPolymeraseorotherDNApolymerasesusing
aThermusthermophilusHB8genomicDNAtemplate,andclonedintosuitableplasmids.Multiplecloneswere
selectedforeachamplificationproductandsubjectedtosequenceanalysis.DNAfragmentsamplifiedusing
CloneAmpHiFiPolymeraseyieldedonly12mismatchedbasesper542,580totalbaseslowerthananalternative
highfidelityenzymefromCompanyA,and10foldlowerthanTaqDNApolymerase.

TipsforamplificationwithInFusionPCRprimers
Thetemplatespecific3'endoftheInFusionPCRprimershouldbe1825ntlong,inordertoensuretemplate
amplification.
TodeterminetheTmofInFusionPCRprimers,useindependentsoftwareforPCRprimeranalysis,such
asOligoAnalyzer3.1fromIDTTechnologies.UsestandardMg2+,Na+,anddNTPconcentrationsusually
recommendedforPCR.
Foroptimalamplification,performtheinitial35PCRcyclesusingtheannealingtemperaturecompatiblewithjust
the3'templatespecificportionoftheInFusionPCRprimer.TheremainingPCRcyclesshoulduseanannealing
temperaturecompatiblewiththeTmoftheentireprimer.
IfyouexperienceinefficientPCRamplification,itmaybenecessarytoredesigntheInFusionPCRprimers,by
eitherextendingorrepositioningthetemplatespecific3'end.

CompatiblePCRpurificationmethods
PCRamplifiedDNAmustbepurifiedpriortotheInFusionCloningreaction.Thiscanbeaccomplishedwithone
ofthefollowingoptions:
NucleoSpinGelandPCRCleanUp
GelextractionenablesselectionofspecificDNAfragmentsofthedesiredsizefrombackgroundPCRbyproducts
orothercontaminants.
ColumnpurificationisappropriateifPCRdidnotproduceabackgroundsmear.
CloningEnhancer(CE)
ThisproprietaryenzymemixremovesbackgroundplasmidDNAandPCRresidue.
CEisappropriateforPCRthatresultsinasinglefragmentoftheexpectedsize,withoutabackgroundsmear.
CEisaconvenienttoolforhighthroughput(HTP)applicationsthatemployhighlyoptimizedPCRcycling
conditionsandprimersthatgeneratecleanDNAfragmentsoftheexpectedsize.
Note:Inmostcases,CEtreatmentdoesnotrequireadditionalcolumnpurificationorgelextraction.However,to
ensurebettercloningresults,PCRlinearizedvectorsmayrequireacombinationofCEtreatmentfollowedbygel
extractiontoseparatealinearizedvectorfrompossiblePCRbyproducts.

TransformationinE.coli
Cellcompetency
InFusionCloningrequiresbacterialcellswithcompetencynolessthan108 cfu/gsupercoiledDNA.

WerecommendStellarCompetentCells(includedinallcurrentInFusionCloningkits).Anygeneralpurpose
cloningE.colistrainshouldbecompatiblewithInFusionCloningaswell.
StellarCompetentCellshavebeenvalidatedforcloningandamplificationoflargevectors(e.g.,BACs,fosmids)
andvectorswithreiteratedsequencessuchasLongTerminalRepeats(LTRs)inretroviral/lentiviralvectors,or
InvertedTerminalRepeats(ITRs)inadenoviralvectors.
TOP10cellsortheirderivatives(e.g.,ccdBSurvival2T1RE.coli),andrelatedstrains(e.g.,DH10B,MC1061)are
suboptimalforInFusionCloning,resultinginalowernumberofrecombinantclones.Thismaybeofparticular
concernifyouareperformingmultiplefragmentcloning,orusingalowcopynumbervector.

StrainsnotrecommendedforInFusionCloning
WedonotrecommendtransformingInFusionreactionmixturesintoanyofthefollowing:
E.colistrainslackingrecA1,orendAmutations
E.colistrainsengineeredforaparticularapplication(e.g.,largescaleproteinexpression)
Grampositivebacterialstrains
BacterialcellscarryingnupG(deoR)mutations
Note:IfitisabsolutelynecessarytouseaparticularbacterialstrainnotvalidatedforInFusionCloning,a1:5
dilutionofthereactionmixmayincreasetransformationefficiency.
TOP10cellsortheirderivatives(e.g.,ccdBSurvival2T1RE.coli),andrelatedstrains(e.g.,DH10B,MC1061)are
suboptimalforInFusionCloning,resultinginalowernumberofrecombinantclones.Thismaybeofparticular
concernifyouareperformingmultiplefragmentcloning,orusingalowcopynumbervector.

Optimaltransformationamounts
Fortransformationofchemicallycompetentbacterialcells(e.g.,StellarCompetentCells),use2.5lofundiluted
InFusionCloningreactionmixper50lofcells.
Fortransformationofelectrocompetentcells,use1lof1:5dilutedInFusionCloningreactionmixper50lof
cells.
(Optional)Forlargertransformationvolumes,5.0lofundiluted,unpurifiedreactionmixcanbetransformedper
100lStellarCompetentCells.
Weadviseagainsttransformingthereactionmixinamountslargerthanthosestatedabove,asitmaybetoxicto
yourcells.

Vectorandinsertpropertiesrelatedtotransformationefficiency
Somevectorsorinsertsmaycontainreiteratedsequences(e.g.,retroviralorlentiviralLTRs,adenoviralITRs,etc.).
Whenworkingwithsuchvectors,itmaybenecessarytooptimizebacterialtransformationtoprevent
rearrangementswithintherecombinantconstructandincreaseitsstabilityduringrescueandamplificationinE.
coli.Followheatshockwithrevivalofthebacteriaat2530Cwhileshakingat120160rpm,andvaryingthe
selectiveantibioticconcentrationand/orgrowthtemperatureoftheplatedtransformationculture(e.g.,25C,30C,
etc.).

SelectedApplications
Cloningageneofinterestinframewithafluorescentprotein(easyprotocol)
1. UseInFusionReadyFluorescentProteinVectors
2. DesignInFusionPCRprimersaccordingtothefollowingresources:pAcGFP1NInFusionReadyvector
information,pAcGFP1CInFusionReadyvectorinformation,orthevectorinformationontheDocumentstabof
theInFusionReadyFluorescentProteinVectorsproductpage.
Note:Topreservetranslationalreadingframecontinuity,yourgeneofinterestshouldnotcontainaSTOP
codonifinsertedupstreamofthefluorescentprotein.Ifthegeneofinterestisdownstreamofthefluorescent
protein,itmayretainitsownSTOPcodon,butthefluorescentproteinshouldnot.
3. AmplifyyourgeneofinterestusingtheInFusionPCRprimersdesignedinStep2.
4. PurifytheamplifiedgeneofinterestusingNucleoSpinGelandPCRCleanUporCloningEnhancer.
5. FollowtheInFusionReadyVectorCloningProtocolAtAGlanceforInFusionreactionandtransformation
instructions.

Cloningageneofinterestinframewithafluorescentprotein(alternativeprotocol)
1. Chooseanyvectorcarryingafluorescentprotein.LinearizeyourvectoreitherbyrestrictiondigestorinversePCR.
2. Purifythelinearizedvectorbygelextractiontoensuretheisolationofonlylinearvectormolecules.
3. DesignInFusionPCRprimersusingtheonlinePrimerDesignTool.(ThePrimerDesignTooliscompatiblewith
MozillaFirefoxorGoogleChromewebbrowsers,butnotwithInternetExplorer.)
Alternatively,instructionsforInFusionprimerdesignaredescribedintheInFusionHDCloningKitUser
ManualandInFusionHDEcoDryCloningKitUserManual.
Regardlessofdesignmethod,werecommendSnapGeneViewerforinsilicoassemblyofyourrecombinant
constructandeasyvisualizationofthelocationsofInFusionprimersinthesenseandantisensestrands.
Notes:Topreservetranslationalreadingframecontinuity,yourgeneofinterestshouldnotcontainaSTOPcodon
ifinsertedupstreamofthefluorescentprotein.Ifthegeneofinterestisdownstreamofthefluorescentprotein,it
mayretainitsownSTOPcodon,butthefluorescentproteinshouldnot.
Ifnecessary,youcanalsoadjustthereadingframecontinuitybyinsertingextranucleotidesbetweenthe
template/genespecificportionandthe15nthomologousportionoftheInFusionPCRprimers,asillustratedin
thetablebelow.(ThecurrentversionofourPrimerDesignTooldoesnotallowforthistypeofadjustment,and
requiresmanualdesignbytheuser.)

5'15nthomologywithvector
sequence

Numberofbasesneededto
maintainreadingframe
0

3'genespecificsequenceofthe
InFusionPCRprimer

1
2

4. AmplifyyourgeneofinterestusingtheInFusionPCRprimersdesignedinStep3.
5. PurifytheamplifiedgeneofinterestusingNucleoSpinGelandPCRCleanUporCloningEnhancer.
6. FollowtheInFusionHDCloningKitUserManualorInFusionHDEcoDryCloningKitUserManualforInFusion
reactionandtransformationinstructions.

Compatibilitywithlargevectors
InFusiontechnologyallowseasycloningofsingleormultipleDNAfragmentsdirectlyintolargevectors(e.g.,
adenoviralvectorsat32.636kb)inasinglereaction,withoutintermediatecloningintotransfer/shuttlevectors.
(PleaseseeFigures1,2,5,andTableIIIoftheAdenoXAdenoviralSystem3Brochurefordetails.)

Compatibilitywithlargeinserts
Thistechnologyhasbeenoptimizedforcloninglargefragments.DNAinsertsupto15kbhavebeensuccessfully
clonedintopUC19usingInFusionCloning.

Tenoutoftencoloniescontainthecorrectinsert(100%efficiency)whencloningfragmentsaslargeas15kb
(resultsconfirmedbycolonyPCRscreening).

Multiplefragmentcloning
Wehavesuccessfullytestedmultiplefragmentcloningwithuptofiveinserts.(SeeFigureandTablebelowfor
cloningschematicandcolonyscreenresults,respectively.)
PrimerdesignformultiplefragmentcloningcanbedonewithouronlinePrimerDesignTool.(ThePrimerDesign
TooliscompatiblewithMozillaFirefoxorGoogleChromewebbrowsers,butnotwithInternetExplorer.)Wealso
haveaPrimerDesignTooltutorialspecificallyformultiplefragmentcloning.
Pleasenotethatbetweentwoadjacentfragments,onlyonehomologousoverlapisrequiredfortheInFusion
reaction.Thisoverlapcanbelocatedoneitherofthefragments,orsplitbetweenthem.

TheInFusionHDCloningSystemhasanimprovedcapabilityforcloningmultiplefragmentsinasingle
reaction.Usingthissystem,cloninguptofour1kbfragmentssimultaneouslyisaseasyascloningasinglefragment.
Thissavesweeksthatwouldotherwisebespentscreeningclonesandsubcloning.

Insert

ColonyScreening

Fragments

Colonies,1/5plated

Correctclones

1kb+1kb

2,128

10/10

1kb+1kb+1kb

83

7/10

1kb+1kb+1kb+1kb

31

8/10

1kb+1kb+1kb+1kb+1kb

14

4/10

Mutagenesis
InFusionCloningallowssingleormultiplebasechanges,deletions,andinsertionsthroughtheuseofinverse
PCR.PleasenotethisapplicationreliesonhighfidelityPCR.ItisessentialtouseaPCRpolymerasewithhigh
proofreadingactivity,suchasCloneAmpHiFiPCRPremix(suppliedwithInFusionHDCloningPluskits).This
polymeraseishighlyrobustandaccurate,enablingamplificationofupto6kbofhumangenomicDNA,10kbofE.
coligenomicDNA,and15kbofLambdaDNA,andexhibitsminimalerrorratesonGCrichtemplates.

MutationfrequencyofCloneAmpHiFiPolymerasecomparedtootherhighfidelityPCRenzymes.Eightarbitrarily
selectedGCrichregionswereamplifiedwithCloneAmpHiFiPolymeraseorotherDNApolymerasesusing
aThermusthermophilusHB8genomicDNAtemplate,andclonedintosuitableplasmids.Multiplecloneswere
selectedforeachamplificationproductandsubjectedtosequenceanalysis.DNAfragmentsamplifiedusing
CloneAmpHiFiPolymeraseyieldedonly12mismatchedbasesper542,580totalbaseslowerthananalternative

highfidelityenzymefromCompanyA,and10foldlowerthanTaqDNApolymerase.
EachPCRprimerdirectsDNAsynthesisintheoppositeorientationoftheotheronacircularvectortemplate.
The3'endsoftheforwardandreversePCRprimersare1825ntthatarecomplementarytothetemplate,ensuring
efficientandspecificamplification.
Mutationsareincorporatedwithinthehomologous15ntoverlaplocatedatthe5'endsoftheforwardandreverse
PCRprimers.(Thishomologousoverlapisrequiredfortherecircularizationofthemutatedvector.)
Singleormultiplebasechanges,deletions,orinsertionscanbeintroducedinasingleInFusionreaction.
Alargerdeletionofanydesirablelengthcanalsobeintroducedbypositioningthe3'endsoftheforwardand
reverseprimersatthebordersitesofadeletion,withhomologousoverhangscarriedbythe5'endofeitherof
theprimers.
TheresultinginversePCRwillgeneratealineardoublestrandedvectorwith5'and3'endscomplementaryto
eachother,andcarryingthe15nthomologousoverlap.(ThisoverlapwillbejoinedthroughtheInFusionreaction
andrecoveryinE.coli,thusgeneratingamutatedvector.)
VectorsamplifiedwithinversePCRmustbetreatedwithCloningEnhancertodestroytheparentalvector.
Additionalpurificationbypreparativegelelectrophoresismayberequiredtoensureisolationofthelinearized
vectorfromPCRbyproductsandpossibleremnantsoftheparentalcircularvector.
Foradditionaldetails,pleaseseeourprerecordedmutagenesiswebinarand/ortheMutagenesiswithInFusion
HDCloningPlustechnote.

CloningshRNA(smallhairpinRNA)
AnshRNAdoublestrandedDNAoligonucleotide(50bp)canbeclonedviaInFusiontechnologyintoa
linearizedshRNAexpressionvector.
ForInFusioncloningofshortsyntheticoligos(between50and150bp),thesuggestedoligotovectormolarratiois
515:1.Dependingontheoligolength,theoptimalratiomustbedeterminedempirically.
Highquality,nonphosphorylatedoligonucleotidespurifiedbydesaltingarecompatiblewithInFusionCloning.
However,3'exonucleaseactivityintheInFusionenzymemixrequiresterminal3'OHgroups.
ClontechoffersawidevarietyofshRNAexpressionvectors.ThesevectorsalsoallowshRNAcloningviarestriction
digestandligation,usingshRNAoligoswithrestrictionsiteoverhangs(generatedbytheOnlineshRNASequence
Designer).
Note:Notallantisenseoligonucleotidesdesignedandtestedfordirectcelltransfection,suchassiRNAs,willbe
equallyefficientwhenexpressedasanshRNAfromavector.ItisusuallyrecommendedtoredesignthesiRNA
oligoforexpressionasanshRNAwithvariousorientationsofthetargetsequenceasasenseorantisensestrand.
Forefficientknockdown,atleastfourdifferentshRNAconstructsaretypicallydesignedandtestedfirstintransient
transfection(usingeasytotransfectcells,ifapplicable),priortoestablishingastablecelllineorrunningin
vivoexperiments.
InordertodistinguishrecombinantshRNAvectors,adiagnosticrestrictionsite(MluI)canbeinsertedintothe
shRNAoligodownstreamfromtheRNAPolymeraseIIITerminationSignal.

CloningamicroRNA(miRNA)precursor
SequencesformicroRNAprecursorsandflankinggenomicDNAcanbeobtainedfromanumberofpublic
databases,includingGenBankandEMBLBank.TheUCSCGenomicBioniformaticsSitehostsaneasyto
navigategenomicdatabasewhichtracksmiRNAs.TheSangerInstitutehostsmiRBase,acompilationofknown
miRNAsequences.
100300bpofDNAflankingthemiRNAprecursorisamplifiedfromgenomicDNAforcloningintothe3'UTRofa
fluorescentprotein,carriedbyanmiRNAexpressionvector.TheflankingDNAensuresefficientprocessingby
Drosha.
ForInFusionCloning,themiRNAprecursor(100300bp)isPCRamplified,incorporating15bpoverhangs
homologoustotheterminiofthemiRNAexpressionvector.Thevectorshouldbelinearizedatthefluorescent
protein's3'UTR.ThesuggestedmiRNAprecursortovectormolarratioforthecloningreactionis35:1,depending
ontheprecursorlength.Optimalmolarratiosmustbedeterminedempirically.
Inthecell,themiRNAprecursoriscoexpressedwiththefluorescentprotein(asdescribedinFigure2ofthistech
note),allowingbothofthefollowing:
Expressionofthefluorescentprotein,resultinginfluorescentcelllabeling.
miRNAprecursorprocessing,resultingintargetedgeneknockdowninfluorescentlylabeledcells.
ClontechoffersTetinduciblemiRNAexpressionsystemsandvectorswitheitheraredorgreenfluorescentprotein
marker.

http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Resources/Cloning_Tips/
In-Fusion_Cloning