InFusionCloningFAQs

ThefollowingFAQsapplytocurrentInFusionCloningkits:InFusionHDCloningPlus,InFusionHDCloning
PlusCE,andInFusionHDEcoDryCloningPlus.

GENERAL
INFO

INSERTS/PRIMER
DESIGN

VECTORS

APPLICATIONS

TIPS

GeneralInformation
WhatisInFusionCloning?
InFusionCloningisahighlyefficient,ligationindependentcloningmethodbasedontheannealingof
complementaryendsofacloninginsertandlinearizedcloningvector.Thistechnologyensureseasy,singlestep
directionalcloningofanygeneofinterestintoanyvectoratanylocus.InFusionconstructsareseamless,
enablingtranslationalreadingframecontinuitywithoutanyinterferingscarsequences.

WhatistheefficiencyofInFusionCloning?
Cloningefficiencyisatleast95%forasingleinsertintoavector.Unliketransformationefficiency,whichismerely
ameasureofthenumberoftransformedcoloniesobtained,cloningefficiencyisameasureofaccuracy,providing
informationonthenumberofcorrectclonesobtainedfromacloningreaction.

HowdoesInFusionCloningwork?
InFusionCloningrequires15bpofoverlapattheterminiofthecloninginsertandlinearizedcloningvector,or
adjacentcloninginsertsifmultipleinsertsaretobejoinedsimultaneously.
These15bphomologousoverlapscanbegeneratedbyPCRamplificationoroligosynthesisofeitherofthe
cloningcomponents.Homologousoverlapsshorterthan12ntorlongerthan21ntarenotrecommended.
Translationalreadingframecontinuityofafusionconstructcanbeadjustedbyaddingnucleotidesbetweenthe
insertspecificsequenceand15ntoverlap.15bpcomplementaryregionsmustbelocatedattheterminiof
adjacentDNAfragmentsortheywillnotbejoinedbyInFusionCloning.
TheInFusionenzymemixgenerates15ntsinglestranded5'overhangsattheterminiofthecloninginsertand
linearizedcloningvector.Theseoverhangsareannealedatthesitesofcomplementarity,andtherecombinant
circularconstructisrescuedinE.coli.(Wedonotrecommenduseofcellswithcompetencylessthan108 cfu/g
supercoiledDNA.)InFusionCloningdoesnotallowforthecovalentassemblyoflinearDNAmolecules.

AbriefoverviewoftheInFusionCloningprotocol.

WhatisthedifferencebetweenInFusionHDCloningPlusandInFusionHDEcoDry
CloningPlus?
InFusionHDCloningPlusincludesliquidInFusionHDEnzymePremix,whereasInFusionHDEcoDryCloning
Plusincludesprealiquoted,lyophilizedInFusionHDEnzymePremix.TheEcoDrykitminimizeshandlingand
storesatroomtemperature.Theliquidcloningreactioniscompletein15min,andtheEcoDrycloningreactionis
completein30min.

WhatisCloningEnhancer(CE)?
CloningEnhancer,orCE,isaproprietaryenzymemixforremovingbackgroundplasmidDNAandPCRresidue,
thuseliminatingtheneedforadditionalpurificationofPCRamplifiedDNApriortotheInFusionreaction.(See
detailsintheInFusionHDCloningKitUserManual.)CEisavailableaspartoftheInFusionHDCloningPlus
CEkitandasaseparateitem.
UseofCEisonlyappropriateifPCRamplificationgeneratesasinglePCRfragmentoftheexpectedsize,without
abackgroundsmear.CEisaconvenienttoolforhighthroughputapplicationsthatemployhighlyoptimizedPCR
cyclingconditionsandprimersthatgeneratecleanDNAfragmentsoftheexpectedsize.
TheadditionofCEtotheInFusionreactionmixisnotrequired,nordoesitincreasecloningefficiencyitsimply
replacesstandardpurificationsteps,providedthatPCRgiveshighqualityresults.

DoestheInFusionCloningmethodintroduceerrorsintothesequence?
Clontechhasnotseenanybaseslippage,baseaddition,orbasedeletionwiththeInFusionCloningenzyme.We
haveclonedandsequencedover4,000separateclonesandvarioushumanopenreadingframessubsequentto

InFusionCloning,andhaverarelyseenanyevidenceoferrorsatthecloningjunctions(<2%).Mostofthe
sequenceerrorsthatwehavecomeacrossareclearlyduetoerrorsinprimersynthesis(thatis,theyappearinall
ormanyoftheclonescontainingaparticularinsert).InFusionCloningisidealformakingerrorfreefusion
constructs.

HowstableistheInFusionCloningenzymemix?
TheInFusionHDcloningenzymemixhasbeenengineeredforincreasedstability,requiresnodilution,andcan
bestoredat20C(liquid)orroomtemperature(EcoDry).

InsertsandPrimerDesign
Whataretherequirementsforahomologousoverlapthatwillfacilitateasuccessful
InFusionCloningreaction?
HomologousoverlapsarenecessaryforInFusionCloning.Appropriatehomologyconsistsofa15ntDNA
sequencecomplementarytothe5'endofalinearizedcloningvectororcloninginsert.Figures2and3in
theInFusionHDCloningKitUserManualorInFusionHDEcoDryCloningKitUserManualprovidedetailed
examples.

HowdoIgeneratehomologousoverlapsbetweentheterminiofcloninginsertsand
linearizedvectors?
HomologousinsertsarecreatedthroughPCRamplificationofcloninginsertsusingprimersspecificallydesigned
toincorporate15ntof5'overhangscomplementarytotheterminiofthelinearizedvector(oradjacentcloning
insert).
Alternatively,15ntofhomologymaybeaddedtoavectorlinearizedviainversePCR,suchthatitoverlapswith
thecloninginsert.Ifsyntheticoligonucleotides(50bp)arebeingcloned,theseoligosmaycarrythe15nt5'
overhangshomologoustotheendsofthelinearizedcloningvectororadjacentDNAfragments.(Highquality,
nonphosphorylatedoligosarecompatiblewithInFusionCloning.)

HowdoIdesignPCRprimerscarrying15ntoverhangscomplementarytotheterminiof
thelinearizedvectororadjacentinsert?
Eachforward(5'3'sensestrand)andreverse(5'3'antisensestrand)PCRprimershouldincludethe
following:
Atemplatespecific(genespecific)portionatits3'end.ToensurespecificandefficientPCRamplification,the
templatespecificportionoftheprimershouldbe1825ntinlength.
15ntofhomologyatthe5'endoftheprimer,complementarytotheterminiofthelinearizedvectororadjacent
inserts(ifmultipleinsertsaretobeclonedsimultaneously).Homologousoverlapsshorterthan12ntandlonger
than21ntarenotrecommended.The15bpcomplementaryregionsmustbelocatedattheterminiofadjacent
DNAfragmentsortheywillnotbejoinedbyInFusionCloning.
(Optional)Toensurecontinuityofthetranslationalreadingframe,ortopreserverestrictionsite(s),additional
nucleotidescanbeaddedtothePCRprimer(s)betweenthetemplatespecificportionandthe15nthomologous
overlap.

WhatistheoptimallengthofthehomologousoverlapbetweentheterminiofthePCR
amplifiedinsertandlinearizedcloningvector?

CurrentInFusionreactionconditionsfavor15bpofhomologousoverlap.Wedonotrecommendusingoverlaps
shorterthan12bporlongerthan21bp.

WhattoolsareavailabletoassistinthedesignofPCRprimerscompatiblewithInFusion
Cloning?
InstructionsfordesigningInFusionPCRprimersareincludedinallInFusionCloningusermanuals.Additionally,
ouronlinePrimerDesignToolfacilitatesprimerdesignforsingleandmultiplefragmentcloning,andis
compatiblewithMozillaFirefoxorGoogleChromewebbrowsers.(InternetExplorerisnotcompatiblewiththe
PrimerDesignTool.)StepbysteptutorialsforthePrimerDesignToolarealsoavailableintheCloning
Resourcessectionofourwebsite.
WealsorecommendSnapGeneViewerasahelpful,freeonlinetoolforinsilicoassemblyofyourrecombinant
construct,manualdesignofInFusionPCRprimers,andadjustmentoftranslationalreadingframecontinuity.

WhyarehomologousoverlapsimportantforInFusionCloningreactions?
ThemechanismforInFusionCloningreactionsemploysa3'exonucleasetogeneratesinglestranded5'
overhangsattheterminioflineardoublestrandedDNA.TheseDNAfragmentsarethenannealedvia
complementary15ntoverlapsattheterminioftheinsert(s)andalinearizedvector.Thevectorcanbelinearized
byinversePCRorrestrictiondigest.Restrictiondigestcanbeperformedwithoneormoreenzymesthatgenerate
5'overhangs(e.g.,EcoRI,BamHI),3'overhangs(e.g.,KpnI),orbluntends(e.g.,HpaII).
ThediagramsbelowshowspecificexamplesoftheInFusionCloningmechanisminaction:

HowdoIcalculatethe15ntoverlapifthevectorislinearizedviarestrictiondigest,
generatinga5'or3'overhang?
The5'overhangofarestrictionsiteisincludedinthe15ntcomplementaryregion.The3'overhangofarestriction
siteisexcludedfromthe15ntcomplementaryregion.Figures2and3intheInFusionHDCloningKitUser
ManualorInFusionHDEcoDryCloningKitUserManualprovidedetailedexamples.
Restrictionsitesusedforvectorlinearizationcanbepreservedintherecombinantvectorbyaddingnucleotidesto
thePCRprimersbetweenthetemplatespecificportionandthe15nthomologousoverlap.TheonlinePrimer
DesignToolallowsyoutochoosewhetherornottopreservetherestrictionsites.(ThePrimerDesignToolis
compatiblewithMozillaFirefoxorGoogleChromewebbrowsers,butnotwithInternetExplorer.)

HowcanIalterthereadingframewhenperformingInFusionCloning?
Thereadingframeisdefinedbytheprimersequence.Forexample,whencreatingafusionprotein,ifthe15bpof
vectorhomologyatthe5'endoftheInFusionPCRprimersequencecorrespondstothelastfivecodonsofthe
vectorreadingframe,youwouldcloneyournewgeneortaginthesamereadingframedownstreamoftheC
terminusofthevectorsequencebyplacingthefirstcodonofthisgenenexttothelastcodonofthehomology
sequence(i.e.,atthe3'end)withoutanyinterferingSTOPcodons.Toshiftthereadingframe,youwouldsimply

addoneortwoadditionalbasesafterthe15bphomologyandbeforethefirstcodonofthetargetgene.For
example:
5'15nthomologywithvector
sequence

Numberofbasesneededto
maintainreadingframe

3'genespecificsequenceofthe
InFusionPCRprimer

0
1
2

HowdoIclonemygeneofinterestinthesametranslationalreadingframeasatag
presentinthecloningvector(e.g.,fluorescentprotein,Myc,HA,etc.)?
Translationalreadingframecontinuitywithatagisadjustedwithinthelengthofthegenespecificportionofthe
PCRprimer,orbyaddingnucleotidesbetweenthegenespecificportionandthe15nthomologyofthePCR
primer.
PleasenotethatthecurrentversionofouronlinePrimerDesignTooldoesnotallowanadjustmentfor
translationalreadingframecontinuity.Assuch,theprimersequenceshouldbemanuallydesignedbytheuser
werecommendSnapGeneViewerasahelpful,freeonlinetooltohelpwiththistask.

CansmallexternalsequencesbeincludedintheInFusionPCRprimer?
Yesexternalnucleotidesequences(e.g.,smalltags,Kozaksequences,restrictionsites,etc.)canbeadded
betweenthetemplate/genespecificportionandthe15nthomologousoverlapoftheInFusionPCRprimer.
OurInFusionWebinarSeriesincludesaprerecordedvideospecifictothisapplication.

ForInFusionCloning,isitaproblemifthe15bpregionofhomologyispresentmore
thanonceinthevector?Willmultiplerecombinationproductsresult?
Internalrecombinationeventsatsitesotherthanthoseadjacenttothevectorlinearizationsiteareextremelyrare.
Therefore,evenifyourdesiredregionofhomologyispresentmorethanonceinthevectorsequence,unwanted
recombinationeventsareunlikelytooccur.

DoIneedtousephosphorylatedPCRprimersforInFusionCloning?
NotheuseofphosphorylatedoligonucleotidesisnotrequiredforInFusionCloning.

WhatoligonucleotidequalityisrequiredforanInFusionPCRprimer?
InFusionPCRprimersshouldbehighqualityoligonucleotides,purifiedbydesalting.GelorHPLCpurificationis
notrequired.

WhatPCRpolymerasesarerecommendedforamplificationoftheInFusioncloning
insert?
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.

DoPCRgenerated3'AoverhangsinterferewithInFusionCloning?
No,3'AoverhangsdonotinterferewiththeInFusionCloningmechanism.

CanIclonemultiplefragmentsintoonevectorinasingleInFusionCloningreaction?
Yeswehavesuccessfullytestedmultiplefragmentcloningwithuptofiveinserts.(SeeFigureandTablebelow
forcloningschematicandcolonyscreenresults,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

CanIsplitthehomologous15ntoverlapbetweentheinsertandvector,oradjacent
inserts?
Yes.Thehomologous15ntoverlapcanbesplitbetweenadjacentDNAfragments.However,splittingtheoverlap
betweenaninsertandvectorcanonlybedoneifthevectorislinearizedviainversePCR.
PrimerdesignforthisoptionisnotfacilitatedbytheonlinePrimerDesignTool.WerecommendSnapGene
Viewerasahelpful,freeonlinetooltohelpwiththistask.
ThediagrambelowshowsInFusionprimerdesignandtheannealingofcomplementarystrands,usinga15nt
overlapsplitbetweenFragment1(red)andFragment2(blue):

DoIhavetopurifythePCRamplifiedinsertand/orvectorpriortoperformingthe
InFusionCloningreaction?
Yes,thePCRamplifiedDNAmustbepurifiedpriortoInFusionCloning.FollowingPCR,verifybyagarosegel
electrophoresisthatyourtargetfragmenthasbeenamplified.Ifasinglebandofthedesiredsizeisobtained,you
caneitherspincolumnpurify(NucleoSpinGelandPCRCleanUp),ortreatyourPCRproductwithCloning
Enhancer(CE).However,ifnonspecificbackgroundormultiplebandsarevisibleonyourgel,isolateyourtarget
fragmentbygelextraction.IfyouusePCRtoamplifyyourvectorandinsertandyouobtainbothaPCRamplified
vectorandPCRamplifiedfragment(s)withoutnonspecificbackground,youcanusetheQuickInFusionCloning
ProtocolprovidedinAppendixAoftheusermanual.

NucleoSpinGelandPCRCleanUp
GelextractionenablesselectionofspecificDNAfragmentsofthedesiredsizefrombackgroundPCRbyproducts
orothercontaminants.
ColumnpurificationisappropriateifPCRdidnotproduceabackgroundsmear.
CloningEnhancer(CE)
ThisproprietaryenzymemixremovesbackgroundplasmidDNAandPCRresidue.
CEisappropriateforPCRthatresultsinasinglefragmentoftheexpectedsize,withoutabackgroundsmear.
CEisaconvenienttoolforHTPapplicationsthatemployhighlyoptimizedPCRcyclingconditionsandprimers
suchthatPCRgeneratescleanDNAfragmentsoftheexpectedsize.

Note:Inmostcases,CEtreatmentdoesnotrequireadditionalcolumnpurificationorgelextraction.However,to
ensurebettercloningresults,PCRlinearizedvectorsmayrequireacombinationofCEtreatmentfollowedbygel
extractiontoseparatealinearizedvectorfrompossiblePCRbyproducts.

WhatisthelargestDNAfragmentcompatiblewithInFusionCloning?
Thistechnologyhasbeenoptimizedforcloninglargefragments.DNAinsertsupto15kbhavebeensuccessfully
clonedintopUC19usingInFusionCloning.

Tenoutoftencoloniescontainthecorrectinsert(100%efficiency)whencloningfragmentsaslargeas15kb
(resultsconfirmedbycolonyPCRscreening).

WhatisthesmallestDNAfragmentcompatiblewithInFusionCloning?
ThesmallestinsertsuccessfullyclonedwithInFusionCloningwasa50bpsyntheticoligonucleotide(including
two15nthomologousoverlapswiththevectortermini).
ForInFusionCloningofshortsyntheticoligos(between50and150bp),thesuggestedoligotovectormolarratio
is515:1.Dependingonoligolength,theoptimalratiomustbedeterminedempirically.
Note:NonphosphorylatedoligonucleotidesarecompatiblewithInFusionCloning.However,3'exonuclease
activityintheInFusionenzymemixrequiresterminal3'OHgroups.

Vectors
WhatcloningvectorsarecompatiblewithInFusionCloning?
AnylinearvectoriscompatiblewithInFusionCloning.Linearizationcanbeaccomplishedinoneofthefollowing
ways:
Restrictiondigestwithoneormorerestrictionenzymes.
ForefficientInFusionCloning,integrityofthelinearizedvectorterminiisessential.Werecommendusinghigh
qualityrestrictionenzymes,andperformingdigestsoverseveralhours.However,overnightrestrictiondigestis
notadvisable.
DephosphorylationofthevectorterminiisnotrequiredthevectorwillnotrecircularizeintheInFusionCloning
reactionmixunlessitcarries15ntcomplementaryoverlapsatitstermini.
InversePCRwithprimerspositionedatthedesiredcloningsite.
Choiceofcloninglocusisflexiblesincesuitablerestrictionsitesarenotrequired.
SimultaneousPCRmediatedmutagenesis(deletion,insertion,basechange)ispossible.(Pleasesee
ourprerecordedwebinaronthisapplication.)
The15bphomologousoverlapscanbeaddedtothePCRlinearizedvector.
PreservetheintegrityofthevectorbackbonebyusingaPCRpolymerasewithhighproofreadingactivity,
likeCloneAmpHiFiPCRPremix(suppliedwithInFusionHDCloningPluskits).Thispolymeraseishighly
robustandaccurate,enablingamplificationofupto6kbofhumangenomicDNA,10kbofE.coligenomicDNA,
or15kbofLambdaDNA.ItiscompatiblewithtwoorthreestepPCRcycling,andexhibitsminimalerrorrates
onGCrichtemplates.

MutationfrequencyofCloneAmpHiFiPolymerasecomparedtootherhighfidelityPCRenzymes.Eight
arbitrarilyselectedGCrichregionswereamplifiedwithCloneAmpHiFiPolymeraseorotherDNApolymerases
usingaThermusthermophilusHB8genomicDNAtemplate,andclonedintosuitableplasmids.Multipleclones
wereselectedforeachamplificationproductandsubjectedtosequenceanalysis.DNAfragmentsamplified
usingCloneAmpHiFiPolymeraseyieldedonly12mismatchedbasesper542,580totalbaseslowerthanan
alternativehighfidelityenzymefromCompanyA,and10foldlowerthanTaqDNApolymerase.
Vectorslinearizedviarestrictiondigestshouldbepurifiedbyapreparativeagarosegel(coveredwithaluminum
foiltopreventDNAdamage).Electrophoresisshouldbedoneatalowvoltagetoensuretheseparationoflinear
andcircular(uncut)vectormolecules.
VectorslinearizedviainversePCRshouldbetreatedwithCloningEnhancer(CE)todestroytheparental
plasmid.CEtreated,PCRlinearizedvectorsmayrequireadditionalpurificationbyagarosegelelectrophoresisif
PCRbyproductsarepresentinthelinearizedvectorprep.

DoesInFusionCloningpreservetherestrictionsite(s)usedtolinearizethevector?
Inordertomaintaintherestrictionsites,nucleotidescanbeaddedtothePCRprimersbetweenthetemplate
specificportionandthe15nthomologousoverlap.
TheonlinePrimerDesignToolallowsyoutochoosewhetherornottopreservetherestrictionsites.(ThePrimer
DesignTooliscompatiblewithMozillaFirefoxorGoogleChromewebbrowsers,butnotwithInternetExplorer.)

DoIhavetodephosphorylatetheterminiofalinearizedvectorforInFusionCloning?
No,dephosphorylationofthevectorterminiisneitherrequirednorrecommendedforInFusionCloning.

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

IsInFusionCloningcompatiblewithvectorscarryingrepeatedsequences?
YesClontechscientistsroutinelyuseInFusionCloningtoclonetransgenesintolentiviralorretroviralvectors
thatcarrylongterminalrepeats(LTRs),aswellasadenoviralvectorsthatcarryinvertedterminalrepeats(ITRs).

Applications
CanIuseInFusionCloningtoassembleacovalentlylinkedlinearDNAmolecule?
No,InFusionCloningdoesnotallowtheassemblyofcovalentlylinkedlinearDNAmolecules.
InFusionCloningkitcomponentsincludealinearizedcloningvector,enablingtherescueofacircular
recombinantconstructinE.coli.

CanIuseacircularcloningvectorforInFusionCloning?
No,circularcloningvectorsarenotcompatiblewithInFusionCloning.Avectormustbelinearizedviarestriction
digestorinversePCR.

CanIuseInFusionCloningtocloneaDNAfragmentgeneratedbyrestrictiondigest?
YesiftheadjacentDNAfragments/oligosorlinearizedvectorcarrythe15bphomologousoverlapsrequiredfor
annealing.15bpoverlapswiththedigestedcloninginsertmaybeaddedtotheterminiofaPCRlinearizedvector
orasyntheticoligonucleotide.

WillInFusiontechnologyallowcloningofaninsertifthesitesofcomplementarityare
locatedatadistancefromthelinearizedvectortermini?
Nohomologous15bpoverlapsshouldbelocatedpreciselyattheterminiofthevectorandinsert.15bp
complementaryregionsnotlocatedattheterminiofadjacentDNAfragmentswillnotbejoinedbyInFusion
Cloning.PCRlinearizationofavectorallowspositioningoftheprimersatthedesiredcloningsite,thusenabling
thegenerationofthe15bpoverlapsatthetermini.

IsInFusionCloningcompatiblewithvectorscarryingrepeatedsequences?
YesClontechscientistsroutinelyuseInFusionCloningtoclonetransgenesintolentiviralorretroviralvectors
thatcarrylongterminalrepeats(LTRs),aswellasadenoviralvectorsthatcarryinvertedterminalrepeats(ITRs).

CanIuseInFusionCloningformutagenesis?
Yes,InFusionCloningallowssingleormultiplebasechanges,deletions,andinsertions.Fordetails,pleasesee
ourprerecordedmutagenesiswebinarand/ortheMutagenesiswithInFusionHDCloningPlustechnote.

CanIcloneanoligonucleotide/shRNAoligonucleotideusingInFusionCloning?
Yes,syntheticoligonucleotides(50bp),carryinghomologousoverlapswiththeterminiofthelinearvector,can
beclonedusingInFusionCloning.Forcloningofshortsyntheticoligos(between50bpand150bp),the
suggestedoligotovectormolarratiois515:1.Dependingontheoligolength,theoptimalmolarratiomustbe
determinedempirically.
Note:NonphosphorylatedoligonucleotidesarecompatiblewithInFusionCloning.However,3'exonuclease
activityintheInFusionenzymemixrequiresterminal3'OHgroups.

CanIuseInFusionCloningtocloneGCrichDNAfragments?
Yes,butspecialconsiderationshouldbegiventothehomologousoverlapsofadjacentDNAfragments.Since
InFusionCloningisbasedontheannealingoftheseoverlaps,itisimportanttotaketheGCcontentintoaccount
forthe15bphomology.WehavenospecificdatashowingvariabilityofcurrentInFusionHDCloningkit
performancedependingontheGCcontentofthe15bpoverlap.However,thefollowingresultswereobtained
usingapreviousversionofthekitInFusionAdvantage:
15bphomologousoverlapswithGCcontentof2040%hadlittleornoeffectontheInFusionAdvantagecloning
efficiency.
15bphomologousoverlapswithGCcontentof6080%showedareducedInFusionAdvantagecloningefficiency
incertaincases.

Tips
ForamoreextensivediscussionofInFusionCloningTips,pleasevisitthispage.

WhataretherecommendedinserttovectormolarratiosforInFusionCloning?
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).

CanImodifythelengthofthehomologousoverlap?Willalongeroverlapimprove
InFusionCloningefficiency?
CurrentInFusionCloningreactionconditionsfavora15bphomologousoverlap.Wedonotrecommendusing
overlapsshorterthan12bporlongerthan21bp.

WillcloningefficiencyincreaseifIusealongerincubationtimefortheInFusionCloning
reaction?
No,anincreaseintheInFusionreactiontimeisnotrecommended.Itmaygenerateunevensinglestranded
regionsattheendsofthecloninginsertandvector,resultingininefficientannealingofthehomologousoverlaps,
thusreducingcloningefficiency.

CanIuseTOP10cellsforInFusionCloning?
TOP10cellsortheirderivatives(e.g.,ccdBSurvival2T1RE.coli),andrelatedstrains(e.g.,DH10B,MC1061)are
suboptimalforInFusioncloning,resultinginalowernumberofrecombinantclones.Thismaybeofparticular
concernifyouareperformingmultiplefragmentcloning,orusingalowcopynumbervector.
WerecommendusingStellarCompetentCells,whichareoptimizedforusewithInFusionCloningandare
includedinallcurrentkits.

WhatbacterialstrainsarecompatiblewithInFusionCloning?
InFusionCloningrequiresbacterialcellswithcompetencynolessthan108 cfu/gsupercoiledDNA.
StellarCompetentCells(includedinallcurrentInFusionCloningkits)aswellasanygeneralpurposecloningE.
colistrainshouldbecompatiblewithInFusionCloning.
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,orusingalowcopyvector.
WedonotrecommendtransformingInFusionreactionmixturesintoanyofthefollowing:
E.colistrainslackingrecA1,orendAmutations
E.colistrainsengineeredforaparticularapplication(e.g.,largescaleproteinexpression)
Grampositivebacterialstrains
BacterialcellscarryingnupG(deoR)mutations

Note:IfitisabsolutelynecessarytouseaparticularbacterialstrainnotvalidatedforInFusionCloning,a1:5
dilutionofthereactionmixmayincreasetransformationefficiency.

CanItransformInFusionCloningreactionmixturesinamountslargerthanwhatis
recommendedintheusermanual?
Wedonotrecommendthis.Transformingthereactionmixtureinanamountlargerthanwhatisstatedintheuser
manualmaybetoxictoyourcells.FortransformationoftheInFusionCloningreaction,use2.5lofundiluted,
unpurifiedreactionmixper50lStellarCompetentCells.
(Optional)Forlargertransformationvolumes,5.0lofundiluted,unpurifiedreactionmixcanbetransformedper
100lStellarComptentCells.

InanInFusionCloningreaction,howmanycoloniesshouldIexpectfromthenegative
control?
Thenegativecontrolprovidedwiththekittypicallyproducesfewerthan5%bluecoloniesthenumberofwhite
coloniesproducedvariesslightlydependingonthestrain.Ingeneral,fewerthan5%ofthewhitecoloniesonan
experimentalplatecontainbackground.Ithasbeenourobservationthat95%ofthecoloniesonexperimental
platesarecorrect.ThisspeakstoInFusiontechnologyshighlevelofcloningefficiency,i.e.,thepercentageof
correctcoloniesrecoveredregardlessofthetotalnumberoftransformedcoloniespresent.

CanIuseelectroporationtotransformtheInFusionCloningreactionmix?
1lof1:5dilutedInFusionCloningreactionmixcanbeelectroporatedinto50lofelectrocompetentbacterial
cells.

HowcanIensuretransformationefficiencyandoverallcloningefficiency?
InFusionHDCloningPluskitsareallinonesolutionsthatmaintainhightransformationefficiencyandalso
providethehighestpossiblelevelofcloningefficiency.Whilehightransformationefficiencyallowsforalarge
numberoftransformedcolonies,highcloningefficiencyspeakstoaccuracyensuringthatover95%of
transformantsarecorrect,thusreducingtheamountoftimenecessarytoscreencolonies.
Theprimersmustbeofgoodqualitytoensurethesequenceofthehomologousregioniscorrect,allowingthe
cloningreactiontoproceedefficientlyandaccurately.
CleanPCRfragmentsarekeyforsuccessfulcloning.WerecommendNucleoSpinGelandPCRCleanUpforyour
purification,whichisincludedinInFusionHDCloningPluskits.
Forcloningefficiency,itisimportantthatthePCRfragmentbepurifiedawayfromdNTPsandPCRprimersafter
amplification.
UsehighlycompetentE.colicellsthathaveatransformationefficiencygreaterthan108 cfu/gsupercoiledDNA.
Mosthomemadecompetentcellsarenotcompetentenough,especiallyifthesecellsarestoredbeforeuse.We
recommendStellarCompetentCells,whichareoptimizedforusewithInFusionCloningandareincludedinall
currentkits.

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