EasySubcloning

Subcloningshouldbeeasyandfast,andworkeverytime.ThefollowingprotocolsminimizethenumberofmanipulationsrequiredtoprepareDNAfragmentsforligations,therebybothsavingtimeandincreasingreliABIlity.

PreparationofDNAfragmentsforligation.

1.Restrictiondigests:

Alwayscutalotofyourstartingplasmidsinasmallvolume;thiswillhelpinthegelpurificationofyourrestrictionfragmentsbygivingyouahighconcentrationofDNAcomparedtoagaroseinyourgelslice..About1µgina20µlreactionisgood.

Fordoubledigests:youalmostneverhavetodigestwithoneenzyme,adjustthebuffer,anddigestwiththesecond.LookintheBiolabscataloguetofindacompromisebuffer,andsaveyourselfsometime.

Don"tusejust1unitofenzymeandwaitanhour:yourtimeisworthtoomuch,andtheenzymesarecheapandpure.Useabouta10-foldenzymeexcessanddigestabout30min.

2.Playingwiththeends.

Blunting5"overhangs:Add1µl2mMallfourdNTPstoyour20µlrestrictiondigest.Add0.5-1µlKlenow(2-5units),andincubateatroomtempfor30min.

Blunting3"overhangs:Add1µl2mMallfourdNTPstoyour20µlrestrictiondigest.Add0.5-1µlT4DNApolymerase,andincubateat37°for5min.T4polymerasehasamoreactive3"to5"exoactivitythanKlenow,andsoispreferredforthisreaction,butKlenowwillwork.

Bluntingbotha3"and5"endinthesamereaction:usingeitherKlenowaloneorT4polymerasealoneoramixturewillworkokay.

Bluntinganend,andthencuttingwithanotherenzymetoproduceanotherendthatissticky:afterbluntingtheendsproducedbythefirstenzyme,add~80µl0.3MNaOACpH5.2,phenol/chloroformextract,chloroformextract,add3volumesEtOH,putat-20°for20min.,spin10minutes,washwith70%EtOH,dry,andresUSPendintheappropriatebufferforthenextrestrictiondigest.Thenaddthesecondrestrictionenzymeandproceed.(Note:I"vetriedjustkillingtheKlenoworT4polymerasebyheatingto70°for20minutes,andthenaddingthesecondrestrictionenzyme.Thishasn"tgivengoodresults,though;IthinkKlenowinparticularissomewhatThermostable).

Phosphatasingtopreventvectorreclosures:Aftertherestrictiondigest,add1µlBiolabscalfintestinealkalinephosphatase.Incubate37°for1hr.(IntheolddaysIusedBoehringerphosphatase:theysellahighconcentrationtypesothatyoucanputin~20unitstohelpphosphatasebluntand3"overhangends,whicharemoreresistanttophosphatasing.Intheolddayswealsousedtochangetoanalkalinebufferforphosphatasing,butthatappearsunnecessary.)

Bluntinganend,andthenphosphatasing:afterblunting,~killtheKlenoworT4polbyincubatingat70°for20min,andphosphataseasusual.ThereisatheoreticalconcernherethattheunusedfreenucleotideswillcompetewiththeDNAendsforthephosphatase,butempiricallyitseemsnottomatter.Ifyou"reworriedaboutthisyoucouldEtOHppt.andresuspendbeforephosphatasing.

CloningTaqpolymerasePCRproductsbyT/Acloning:Taqpolymerasehasanefficientterminaltransferaseactivitythataddsasinglenucleotidetothe3"endofbluntendedduplexDNA.ThisterminaltransferaseactivitygreatlyprefersaddingAoveraddingC,G,orT,somostPCRproductswillhavesingle3"Aoverhangs.Thesecanbeclonedefficientlyintoavectorcontainingsingle3"Toverhangsonitsends.Youcouldbuykitscontainingpreparedvectorsofthisstructure,butyoucaneasilyproducetheequivalentyourself.Aftercutting1µgofanyvectorwithabluntcutter(e.g.Bluescript,usingEcoRV)dilutethedigestwith1volumeof1XPCRbuffer,adddTTPto0.1mM,add2UTaqpolymerase,andincubateat72°for20minutes.TheTaqwilladdasingleTtothe3"endsofthevectorDNA.Thevectorwillnowself-ligateveryinefficiently,andshouldacceptTaqgeneratedPCRproducts.Inpractice,however,ligationsofsuchvectorswithPCRfragmentsyieldonly~10-50%cloneswithinserts,soitisbesttouseblue/whiteselectiontoidentifycolonieswithinserts;almostallthewhitecolonieswillhaveaninsert.

3.GelpurifyingDNAfragments:IgenerallygelpurifyallDNAfragments;evenifI"mjustlinearizingavectorIrunitoutonageltopurifyitawayfromtheenzymesandbuffersitisin.GelpurificationprovidesaquickwaytopurifytheDNA,andallowsyoutocheckwhatyourputtingintoyourligationtomakesureit"salright.

A.Usealowmeltagarosegel,runinfresh1XTAEinadecentlycleangelbox.UseSeaplaqueagarosefromFMC.Formostpurposes,usea0.8%gel.Ifyouneedgoodresolutionunder500bp,youcangoupto2%.NusieveagarosefromFMCalsoworksforcloning,andcanresolvefragmentsdownto50bporless.Post-stainingthegelwastestime,sorunthegelwithethidiuminit.Include1µlof10mg/mlethidiumbromideper50mlofgel.Youdon"tneedtoaddEtBrtotherunningbufferunlessyouarerunningthegelverylongandarelookingatsmallfragments(inwhichcasetheEtBrwillrunoutofthebottomofthegelandscrewyouupunlessyouaddittotherunningbuffer).Iload1µgofDNAina3.5mmwidewell.

B.Don"twasteyourtimebyrunningthegelslowly.Useabout5voltspercm,i.e.100Vforaminigelbox,150Vforamediumgelbox.

C.Whenthegelisdone,placeitonacleanpieceofsaranwraponalongwave(365nm)UVboxandtakeapicture.MakesuretouselongwaveUVandminimizetheexposuretoUVtoavoiddamagingtheDNA.Cutthedesiredbandsoutofthegelwithacleanscalpel.Turnthegelsliceonitssideandtrimofftheextraagarose.PlacethegelsliceinanEppendorftube.

D.Spinthegelslicedowntothebottomofthetube,andplaceina70°tempblocktomelt.Pipettethemeltedagaroseup/downtogetauniformmixture.Totalvolumeofthegelsliceshouldbe~40µl.Canfreezethegelslicetostore.

Ligation

Add2µl10Xligasebuffertoatube(thecommercialNEBstuffisfine).Add2µlwater.Meltyourgelslicesina70°waterbath.Add10µlofyourinsertgelslice,5µlofyourvectorgelslice.Wait~30secformixturetocool,add1µlofT4DNAligase,mixbypipettingup/down,andimmediatelyplaceonicetocool.Alwaysdoanoinsertcontrol,inwhichyoureplacetheinsertgelslicewith10µlwater.Incubatetheligationovernightat16°,oratroomtemp.for~3hours.

10XLigasebuffer

0.5MTrispH7.8

0.1MMgCl2

50mMDTT

2mMEDTA

4mMrATP

Transformation

A.Melttheligationmixina70°tempblock.Add80µl0.1MTris,pH7.4,andplaceonicetocool.Add100µlcompetentcells,andtransformusingtheprotocolinthismanual.Thereisalsoaprotocolforquickplasmidminipreps,andlargescalepreps.

B.Choiceofhoststrainfortransformation:Strainsgenerallyusedforcloninghavemutationsthatsuppressrecombination,theideabeingthatthishelpsavoidgettingrearrangedclones.Inpractice,unlessyourplasmidcontainsarepeatedsequence,thefrequencyofsuchrearrangementsissolowastonotbeworthworryingabout.Therec-mutations,however,maketheE.colistrainssick,andasaconsequenceyourtransformantsgrowupslowly.Therefore,IrecommendusingahealthyE.colistrainforroutinecloning.ThestrainTG1hasthefollowinggenotype:supEhsd5thi(lac-proAB)F"[traD36proAB+lacI9lacZM15].TG1coloniesgrowupin~8hr.Thecoloniescanbepickedandgrownupto2mlculturesforminiprepsin5hours.Reallytiny8hourcoloniesaremosteasilypickedunderadissectingscopeusingastraightplatinumwire.Becauseitgrowssofast,youcanoftensaveadaybyusingTG1ratherthanalesshealthystrain.TG1canbeusedforblue/whiteselectionwithpUCanbluescriptvectors:whengrowingthestrainuptomakecompetentcells,startwithacolonyfromaminimalplate(thisselectsforcellscarryingtheF"episome,whichcarriesthenecessarylacZconstruct).Ittakeslongerthan8hoursforthebluecolortodevelop,(~11hours),soyouhavetowaitalittlelongertopickthecoloniesifyou"reusingthis.Trylookingattheplatesagainstblackandwhitebackgroundstohelpseethecolorwhenitisjuststartingtodevelop.BluescriptSKandpUCvectorsgivegoodcolordevelopment.BluescriptKStakesmuchlongertodevelopthebluecolor,sodon"tusethisvectorunlessyouhaveto.ForDH5alphaplatedintopagar,usingblue/whiteselectionwithBSSK,gettinycoloniesat12hours,pickablecoloniesat14hours.At14hours,thebluecolorisjustbarelyvisIBLe.

TouseordinaryampplateswithanIPTG/XGALoverlayforblue/whiteselection:

1.Myoldmethod:Taketheampplatesoutofthecoldroomtowarmuptoroomtemp.duringthetransformation.MeltsomeLsoftagarinthemicrowave,andaliquot3mlintosterileglasstubes,andplaceina50°temp.block.Toplateatransformationmix:justbeforeuse,add40µl24mg/mlIPTG,40µl20mg/mlXgalinDMF,10µl20mg/mlamp(orcarb)(storeallthesestocksseparatelyat-20°;youcanmixthestockstogetherimmediatelybeforeusetomakeapremix)tothe3mloftopagar,addthetransformationmix,quicklyvortexandpourontotheampplate.Coloniestakelongertogrowupembeddedintopagarthanonanordinaryplate.Somecolonieswillburstuptothesurfaceandbecomemuchlargerthantheothers.Afterthecoloniesareevident,youcanputtheplatesat4°andthebluecolorwillcontinuedeveloping.

2.Mynewmethod,adaptedfromEwaDavison.Makeup2%X-galinDMF,and24mg/mlIPTGinwater,asinmethod#1.Mixthesetwosolutions1:1,andspread100µlofthemixtureonanordinaryAmpplate.Lettheplatesitatleastonehourtoallowtheliquidtoabsorbintotheagar.Thenplatethetransformationmixasperusual.TheIPTG/Xgalthathasdiffusedintotheplatewillturntheappropriatecoloniesblue.

Commentary

A.Don"tbeawimpaboutmulti-stepconstructs.Theturnaroundtimeonclonescanbeaslittleas24hourswiththeaboveprotocols.Makeliberaluseofpartialdigestsandtripleligationstohelpcutdownonthenumberofstepsrequired;theseworkjustfine.

B.Peoplewhocomplainthatbluntendligationsortripleligationsdon"tworkwellaregenerallyinexperiencedclonerswhoaretryingtoplacetheblamethatreallylieswithothermistakestheymadeintheprocedure;inmyhandstheseligationswork~100%ofthetime.PeoplealsocomplainthatdoingtheligationintheagarosehasalowerefficiencythanyouwouldgetifyoupurifiedtheDNAfragmentsfirst(e.g.byß-agarasedigestionandethanolprecipitation,orusingglassbeadpurification).Whilethismaybetrueintheory,inpracticeIgetmyclone~100%ofthetimedoingtheligationsinagarose(samplesize=severalhundredclonings),soitseemspointlesstoincreasetheefficiencyanymore.Thepurificationprocedurescantakeseveralhours,andcarrytheriskofcatastrophicfailure:youcouldlosetheDNApelletandnotrealizeit"smissinguntilyourcloningfails.Ifoundthatwhenteachingnovicestoclone,theyhavethehighestsuccessratewhenusingaprocedurethatminimizesthenumberofstepstheymightscrewup.

C.Partialdigests:Setup8tubescontaining2µgplasmidin20µlrestrictionbuffer.Add8unitsenzymetothefirsttube.Tranfer10µlfromthefirsttubetothesecond,mix,transfer10µlfromthesecondtubetothethird,mix,andsoon.Finallytransfer10µlfromthelasttubetothefirstone.Incubateallthetubesat37°for1hour,andheatkilltheenzymeat70°for10min.Becausethepatternsfrompartialdigestscanbecomplex,itisoftennecessarytorunquitealongagarosegeltoresolvealltheproducts.Becareful,becausetwoofthebandsthatshowupwillbesupercoiledandnickedplasmid;don"tconfusethesewithlinearfragments.Also,ifyourfragmentrunsnearthesupercoiledornickedplasmidandgetscontaminatedwithevenasmallamountofeitherofthese,youwillgetbackyourstartingcloneatahighfrequencyfromthetransformation.Tocontrolforthis,doanoligasecontrolligation,andseeifitgivesyoucolonies.

D.Tripleligationsworkgreataslongaseachofthethreejointsbeingmadehasadifferenttypeofend,sothattheorientationoftheligationsareforcedandonlyonetypeoftripleligationproductispossible.

Miscellaneous

TheenzymeSma1doesn"talwaysleaveabluntendasadvertised.Therefore,becarefulaboutusingthissitewhentryingtopreserveanopenreADIngframe.