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11/12/2016

Addgene:PlasmidCloningbyRestrictionEnzymeDigest(withProtocols)

PlasmidCloningbyRestrictionEnzymeDigest(akaSubcloning)
Summary

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ThefollowingtechniquecanbeusedtoeasilymoveanypieceofDNAfromonevectortoanotheraslongasitis
alreadyboundedbyrestrictionsitesthatarealsopresentinthesameorientationonyourtargetvector.Ifyouare
notsurewhatvectortouse,youcancheckoutourEmptyBackboneReference.

RestrictionDigestof
PlasmidDNA

Background
Subcloningbyrestrictiondigestisacommonlyusedlabtechnique.Forthepurposesofthistutorialwewilldiscuss
howtomoveacDNAfromoneplasmidtoanother.However,thesametechniquecanbeusedtomove
promoters,selectablemarkers,oranyotherDNAelementbetweenplasmids.

DNALigation
BacterialTransformation

Let'sassumethatyouarebeginninganewprojectonyourgeneofinterest(YGOIforshort).YoumightneedtoexpressYGOIincultured
mammaliancells.TheproblemisthattheonlyversionoffulllengthcDNAyoucanfindforYGOIisinabacterialexpressionvector.Usingsubcloning,
youcaneasilymoveYGOIintoamammalianexpressionvector.

Design(Choosingenzymes)
ManyDNAanalysistools,includingAddgenesSequenceAnalyzer,allowyoutoidentifywhichrestrictionsitesarepresentinagivensequence.
Whenselectingrestrictionenzymes,youwanttochooseenzymesthat:
Flankyourinsert,butdonotcutwithinyourinsert
Areinthedesiredlocationinyourrecipientplasmid(usuallyintheMultipleCloningSite(MCS)),butdonotcutelsewhereontheplasmid
Willresultinyourinsertbeinginthecorrectorientationintherecipientplasmid.(Youdon'twanttoexpresstheantisenseversionofyour
gene!)
Areinframewithtagsorfusionproteinsintherecipientplasmid(ifyouarecreatingafusionprotein)
Ideally,youwillfindtwodifferentrestrictionenzymesforyoursubcloning.Itisalsopossibletouseasingleenzyme,butthiswillrequirephosphatase
treatmentofyourrecipientplasmidaswellasaspecificallydesignedtestdigestlatertoverifythattheinsertwasclonedinthecorrectorientation.
Ifyoucannotfindenzymesthatmeetthesecriteria,donotfear.Youhaveotheroptions,suchas:
Addingdesiredrestrictionsitestoflankyourinsert:YoucanusePCRBasedCloningandaddrestrictionsitestotheendsofyouroligos.This
willallowyoutoproduceaversionofyourinsertflankedbyrestrictionsitescompatiblewiththerecipientplasmid'sMCS.However,youstill
needtoavoidrestrictionenzymesthatcutwithinyourinsert.
Addingdesiredrestrictionsitestoyourrecipientplasmid:YoucanmodifytheMCSofyourrecipientplasmidusingAnnealedoligoCloning.

http://www.addgene.org/plasmidprotocols/subcloning/

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Addgene:PlasmidCloningbyRestrictionEnzymeDigest(withProtocols)

Ifyouareluckyenoughtohavemultipleoptionsforenzymesthatflankyourinsertandwillresultincorrectorientationintherecipientplasmid,itis
usefultoseeifonesetofenzymeswillworkinthesamerestrictionenzymebuffer(seeNewEnglandBiolabsformoreinformationaboutrestriction
enzymebuffers).Ifyouselectenzymesthatcanfunctioninthesamebuffer,itwillsaveyoutimeinfuturesteps.

ExperimentalProcedure

DigestyourDNA:
Setuprestrictiondigestsforyourdonorandrecipientplasmids.BecauseyoulosesomeDNAduringthegelpurificationstep,itisimportanttodigest
plentyofstartingmaterial.Werecommend1.52gofdonorplasmidand1gofrecipientplasmid.Itisalsocriticalthatasmuchoftherecipient
plasmidaspossiblebecutwithbothenzymes,andthereforeitisimportantthatthedigestgoatleast4hoursandaslongasovernight.
Ifyouaregoingtouseonlyonerestrictionenzyme,orenzymesthathavecompatibleoverhangsornooverhangsafterdigestion,youwillneedto
useaphosphatasetopreventrecircularizationoftherecipientplasmid.Youshouldtreatyourdigestedrecipientplasmidwithaphosphatasepriorto
theligationsteporpriortothegelpurificationstep,dependingonthephosphataseyouchoose.CIP(calfalkalinephosphatase)orSAP(shrimp
alkalinephosphatase)arecommonlyused.Followthemanufacturersinstructions.

Isolateyourinsertandvectorbygelpurification:
RunyourdigestedDNAonanagarosegelandconductagelpurificationtoisolatetheDNA.Whenrunningagelforpurificationpurposesitis
importanttohavenicecrispbandsandtohavespacetocutoutthebands.Becauseofthiswerecommendthatyouuseawidegelcomb,runthe
gelontheslowerside,andskiplanesbetweensamples.InadditiontoaDNAladderstandard,itisalsoagoodideatorunanuncutsampleofeach
plasmidtohelpwithtroubleshootingifyourdigestsdontlookasyouexpected.
Onceyouhavecutoutandpurifiedyourinsertandrecipientplasmidbackbonebandsawayfromthegelviayourfavoritegelpurificationmethod,itis
importanttodeterminetheconcentrationofrecoveredDNA.

Ligateyourinsertintoyourvector:
ConductaDNALigationtofuseyourinserttoyourrecipientplasmid.
Werecommendaround100ngoftotalDNAinastandardligationreaction.Youideallywantarecipientplasmidtoinsertratioofapproximately1:3.
Sincethenumberofbasepairsforeachvaries,itisdifficulttocalculatethisbasedonDNAconcentrationalone.Onemethodistoconduct2ligations
foreachplasmidyouaretryingtocreate,withvaryingratiosofrecipientplasmidtoinsert.
Itisalsoimportanttosetupnegativecontrolsinparallel.Forinstance,aligationoftherecipientplasmidDNAwithoutanyinsertwilltellyouhow
muchbackgroundyouhaveofuncutorselfligatingrecipientplasmidbackbone.

Transformation:
Transformyourligationreactionintoyourbacterialstrainofchoice.Followthemanufacturersinstructionsforyourcompetentcells.
Formoststandardcloning,youcantransform12lofyourligationreactionintocompetentcellssuchasDH5alphaorTOP10.Ifusingmuchless
totalDNA(<1ng)orifyouarehavingtroublegettingcolonies,youmightwanttousehighercompetencycells.Additionally,ifyourfinalproductis
goingtobeverylarge(>10kb)youmightwanttouseelectrocompetentcellsinsteadofthemorecommonchemicallycompetentcells.
Thenumberofbacterialcoloniesresultingfromyourtransformationwillgiveyouthefirstindicationastowhetheryourtransformationworked.Your
recipientplasmid+insertplateshouldhavesignificantlymorecoloniesthantherecipientplasmidaloneplate.Therecipientplasmidalonecontrolwill
tellyouyourbackgroundlevelormorespecificallyitwilltellyouhowmanycoloniesyoucanexpectonyourrecipientplasmid+insertplatethatare
notcorrect.
Ifyouhaveahighnumberofcoloniesonyourrecipientplasmidaloneplate,youcantryligatingtherecipientplasmidaloneinthepresenceand
absenceofligase.Ifthecoloniesarearesultofuncutemptyplasmid,youwillstillhavecolonieswhenyoudonotaddligase.Ifthecoloniesarea
resultofrecipientplasmidselfligation,youwillseesignificantlymorecolonieswhenyouaddligase.
Ifyoudonotseeanycolonies,youshouldconductapositivecontroltoensurethatyourtransformationworked.Youshouldalsoverifythatyouare
platingontheappropriateantibioticandtryvaryingtheamountofrecipientplasmidtoinsertintheligationreaction.
http://www.addgene.org/plasmidprotocols/subcloning/

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Addgene:PlasmidCloningbyRestrictionEnzymeDigest(withProtocols)

IsolatetheFinishedPlasmid:
Finally,youwillneedtopickindividualbacterialcoloniesandcheckthemforsuccessfulligations.Pick310coloniesdependingonthenumberof
backgroundcoloniesonyourcontrolplate(themorebackground,themorecoloniesyouwillneedtopick)andgrowovernightculturesforDNA
purification.
AfterpurifyingtheDNA,conductadiagnosticrestrictiondigestof100300ngofyourpurifiedDNAwiththeenzymesyouusedforthecloning.Run
yourdigestonanagarosegel.Youshouldseetwobands,onethesizeofyourvectorandonethesizeofyournewinsert.Ifyouusedonlyone
enzymeorusedenzymeswithcompatibleoverhangsyouwillneedtoverifytheorientationofyourinsert,soyoumaywanttodesignadiagnostic
digestforthispurpose.

Congratulations,younowhaveYGOIinamammalianexpressionvectorandcanbeginyourstudies.
ReferencePage|Top|Index

http://www.addgene.org/plasmidprotocols/subcloning/

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