TAIL PCR Protocol

TAILisaseriesofreactionsthatareintendedtomapwhereaT-DNA(transferDNA)hasinsertedwithinthegenome.Themaincomponentsofthe3reactionsaretheAD(ArbitraryDegenerate)primers,borderprimers,andDNAfromtheT-DNAlinesthataretobemapped.ADprimersaredegenerateprimersthatannealthroughoutthegenome.TheborderprimersarespecificfortheleftandrightbordersoftheT-DNA.Fromtheprimaryreactiontothetertiary,theborderprimersgetclosertotheedgeoftheT-DNA.Thatiswhya""shift""isvisIBLewhenrunningagelwiththesecondaryandtertiaryreactionsnexttoeachother.ThesuccessrateofTAIL-PCRvaries,dependingonhowmanyDNAsamples,ADprimers,andborderprimersareused.1.DilutetheDNAsample1:5(DilutemoreorlessdependingonDNAconcentration.)2.Add5µLDNA,and5µLADprimerstoPCRplateaccordingtothediagrambelow(eachADprimerhasaspecificconcentration,seeAdditionalInformationattheendoftheprotocol):NOTE:Keepplateonicethroughouttheprocedure.

DNA1AD1	DNA1AD2	DNA1AD3	DNA1AD4	DNA1AD5	DNA1AD6	DNA1AD1	DNA1AD2	DNA1AD3	DNA1AD4	DNA1AD5	DNA1AD6
DNA2AD1	DNA2AD2	DNA2AD3	DNA2AD4	DNA2AD5	DNA2AD6	DNA2AD1	DNA2AD2	DNA2AD3	DNA2AD4	DNA2AD5	DNA2AD6
DNA3AD1	DNA3AD2	DNA3AD3	DNA3AD4	DNA3AD5	DNA3AD6	DNA3AD1	DNA3AD2	DNA3AD3	DNA3AD4	DNA3AD5	DNA3AD6
DNA4AD1	DNA4AD2	DNA4AD3	DNA4AD4	DNA4AD5	DNA4AD6	DNA4AD1	DNA4AD2	DNA4AD3	DNA4AD4	DNA4AD5	DNA4AD6
DNA5AD1	DNA5AD2	DNA5AD3	DNA5AD4	DNA5AD5	DNA5AD6	DNA5AD1	DNA5AD2	DNA5AD3	DNA5AD4	DNA5AD5	DNA5AD6
DNA6AD1	DNA6AD2	DNA6AD3	DNA6AD4	DNA6AD5	DNA6AD6	DNA6AD1	DNA6AD2	DNA6AD3	DNA6AD4	DNA6AD5	DNA6AD6
DNA7AD1	DNA7AD2	DNA7AD3	DNA7AD4	DNA7AD5	DNA7AD6	DNA7AD1	DNA7AD2	DNA7AD3	DNA7AD4	DNA7AD5	DNA7AD6
DNA8AD1	DNA8AD2	DNA8AD3	DNA8AD4	DNA8AD5	DNA8AD6	DNA8AD1	DNA8AD2	DNA8AD3	DNA8AD4	DNA8AD5	DNA8AD6

Key:DNA1,DNA2,DNA3,...=IndividualDNAsamplesforT-DNAmapping.Add5µLDNA(1°reaction)inanentirehorizontalrow(e.g.A)foreachindividual.AD1,AD2,AD3,...=ArbitraryDegenerateprimers.Add5µofthe4XADprimer(1°reaction)toeachverticalcolumnasdiagramindicates.lightyellow=Lefthalfofplate-AddLB1primercocktail.grey=Righthalfofplates-AddRB1primercocktail.3.Startthe1°Reaction(detailedinAdditionalInformation)programonthermalcyclerandpressPAUSE,lettingtheblockcoolto4°C.4.MixtheLB1andRB1cocktailsaccordingtoTAILRecipespreadsheetincluded.NOTE:AddTaqpolymeraselast.5.Add10µLofeachcocktail(LB1andRB1)toappropriatewellsaccordingtopreviousdiagram.6.PlaceplateinthermalcyclerandpressPAUSE,againtoallowthereactiontoproceed.7.Topreparethe2°reaction,dilute1°TAILreaction200-foldbytransferring1µLPCRproductsto199µLddH2O.(Thisismosteasilyachievedthroughtheuseofamulti-channelPipette.)8.Setup2°reactionplateaccordingtosamediagram,exceptuse4µLdilutedDNA.NOTE:Asbefore,keepplateonicethroughoutpreparation.9.Add5µLoftheADprimerstotheappropriatewells.10.Start2°ree;reactionprogramonthermalcyclerandpressPAUSE.11.Add11µLofborder(LB2orRB2)cocktailtoappropriatewellsandplaceplateinthermalcycler.PressPAUSEtoallowreactiontoproceed.12.Oncethe2°reactionhascompleted,theproductscaneitherbesequencedora3°reactioncanberuntofurtherpurifythePCRproductsiftherearemanynonspecificproducts.CONTINUEifa3°reactionisneeded.Topreparesamplesforsequencing,SKIPtostep25.13.The3°reactionispreparedlikethe2°needstobediluted100-foldandtheoverallreactionvolumeis50µL.Addthedilutedproductsfromthe2°reactiontoanewPCRplate.Again,keepreactiononiceanduseamulti-channelpipettefordiluting.14.Add12.5µLoftheADprimerstotheappropriatewells.15.Startthe3°reactionprogramonthethermalcyclerandpressPAUSE.16.MixtheLB3andRB3cocktail(addingtheTaqlast)andadd32.5µLtoappropriatehalfofplate.17.Flashspininatabletopcentrifugetoassureallreactioncontentsareatthebottomofthewells.18.PlaceplateinthermalcyclerandpressPAUSEagaintoallowreactiontoproceed.19.Tosequenceentirecontentsofplate,SKIPtostep25.Torunagelandvisualizethe3°ree;reactions,gollowthesesteps:Preparealarge1%agarosegelwith4rowsof50wells(200totalwells).20.Addtheappropriateladder(100bpor1kb)tothefirstandlastwellineachrow.21.Usingamulti-channelpipette,draw7µLfromrowAorthe2°reaction.ExpelthisamountonapieceofParafilm.Usingthesamepipettetips,draw3µLofloADIngdyeandaddittothedropletsontheparafilm.Mixthedyeandreactioncontentsbypipettingupanddown.22.Withoutchangingtips,drawall10µLofthesamplesandaddthemtothegelstartingnexttotheladderinthetop,leftportionofthegel.NOTE:Usingthemulti-channelpipettewillleaveaspacebetweenthesamples,thisisdesired.23.Discardthepipettetipsandrepeatpreviousstepuntilentire2°reactioncontentsareloadedintothegel.Assureaspaceisleftbetweenall2°reactionsaddedtogel.24.Now,dothesamewiththe3°reactions,addthe10µLofthe3°reactionsdirectlynexttothe2°reactions.Ifloadedproperly,alllaneswillbefilledwithoutspaces.Thiswillmakethegeleasiertoanalyze.Thereshouldbeavisibleshiftinproductlengthfromthe2°tothe3°raction.Iftherearemultiplebandsvisibleinonelane,purifyindividualbandsforsequencingviatheTopoCloningProcedure.Ifsinglebandsexistinthe3°reaction,continuetostep25forproductpurification.25.ThePCRproductsmustbepurifiedbeforetheycanbesquenced.ThiscanbedoneindividuallyviatheQiagenPCRpurificationprotocolorenzymepurifiedasexplainedinthisprotocol.Transfer5µ:of2°reactionPCRproductstoanewplate.(Again,thisisveryeasywithamulti-channelpipette).26.MixtheEnzyme(Exol/SAP)Purificationcocktailasfollows:

ddH2OExoISAP(PCRProducts)TOTAL

1.4µL0.2µL0.4µL---(5.0µL)2.0µL(7.0µL)

27.Add2.0µLenzymepurificationcocktailtoDNAsamples(onice).Flashspinplateinatabletopcentrifuge.28.Runreactioninthermalcycler.Usefollowingprogram:Step1=37°Cfor20min.Step2=80°Cfor15min.Step3=4°CforeverStep4=END**Thereactionsarenowreadytobesequencedwiththecorrespondingborderprimers.ADDITIONALINFORMATIONTAIL1°REACTIONPROGRAM:

ControlMethod:CALCULATED

1=4°for2min.

2=93°for1min.

3=95°for1min.

4=94°for30sec.

5=62°for1min.

6=72°for2min.30sec.

7=Gotostep4for4morecycles

8=94°for30sec.

9=25°for3min.

10=Rampfor72°at0.2°/sec,72°for2min.30sec.

11=94°for10sec.

12=68°for1min.

13=72°for2min.30sec.

14=94°for10sec.

15=68°for1min.

16=72°for2min.30sec.

17=94°for10sec.

18=44°for1min.

19=72°for2min.30sec.

20=Gotostep12,for14morecycles

21=72°for5min.

22=4°forever

23=ENDTAIL2°REACTIONPROGRAM:

ControlMethod:CALCULATED

1=4°for2min.

2=94°for10sec.

3=64°for1min.

4=72°for2min.30sec.

5=94°for10sec.

6=64°for1min.

7=72°for2min.30sec.

8=94°for10sec.

9=44°for1min.

10=72°for2min.30sec.

11=Gotostep2,for11morecycles

12=72°for5min.

13=4°forever

14=ENDTAIL3°REACTIONPROGRAM:

ControlMethod:CALCULATED

1=4°for2min.

2=94°for10sec.

3=44°for1min.

4=72°for2min.30sec.

5=Gotostep2,for19morecycles

6=72°for5min.

7=4°forever

8=ENDAD(ArbitraryDegenerate)PrimerSequencesandConcentrations:

AD1:NGTCGASWGANAWGAAAD2:TGWGNAGSANCASAGAAD3:AGWGNAGWANCAWAGGAD4:STTGNTASTNCTNTGCAD5:NTCGASTWTSGWGTTAD6:WGTGNAGWANCANAGA

128-folddegenerate12µM128-folddegenerate12µM128-folddegenerate12µM256-folddegenerate16µM64-folddegenerate8µM256-folddegenerate16µM

StockconcentrationsofADprimersshouldbe20µM.ToachievetheconcentrationsrequiredforTAILreactions,diluteinaseperatetube.Thefinalamountof400µLissufficientforall3TAILreactions.64-folddegenerateAdd160µLprimerand240µLddH2O128-folddegenerateAdd240µLprimerand160µLddH2O256-folddegenerateAdd320µLprimerand180µLddH2OAlternatePlateSetupsforTAILIfcertainADprimersand/orborderprimersarefoundtoproducemorereliableproductsthereisnoneedtousetheotherprimers.Asanexample,IfoundtheLBprimertoworkmoreoftenthantheRBprimer.Similarly,IfoundtheAD1andAD4primerstogeneratenonspecific(vector)productsatahighrate.Therefore,IdesignedaplateusingONLYtheLBprimerandAD2,AD3,AD5,andAD6primers.ThisincreasedthemaximumamountofDNAsamplesthatIcouldrunononeplatefrom8to24.Obviouslythiscansavealotoftimeandmaterials.Hereisanexampleofthemodifiedplatesetup.

DNA1AD2	DNA1AD3	DNA1AD5	DNA1AD6	DNA9AD2	DNA9AD3	DNA9AD5	DNA9AD6	DNA17AD2	DNA17AD3	DNA17AD5	DNA17AD6
DNA2AD2	DNA2AD3	DNA2AD5	DNA2AD6	DNA10AD2	DNA10AD3	DNA10AD5	DNA10AD6	DNA18AD2	DNA18AD3	DNA18AD5	DNA18AD6
DNA3AD2	DNA3AD3	DNA3AD5	DNA3AD6	DNA11AD2	DNA11AD3	DNA11AD5	DNA11AD6	DNA19AD2	DNA19AD3	DNA19AD5	DNA19AD6
DNA4AD2	DNA4AD3	DNA4AD5	DNA4AD6	DNA12AD2	DNA12AD3	DNA12AD5	DNA12AD6	DNA20AD2	DNA20AD3	DNA20AD5	DNA20AD6
DNA5AD2	DNA5AD3	DNA5AD5	DNA5AD6	DNA13AD2	DNA13AD3	DNA13AD5	DNA13AD6	DNA21AD2	DNA21AD3	DNA21AD5	DNA21AD6
DNA6AD2	DNA6AD3	DNA6AD5	DNA6AD6	DNA14AD2	DNA14AD3	DNA14AD5	DNA14AD6	DNA22AD2	DNA22AD3	DNA22AD5	DNA22AD6
DNA7AD2	DNA7AD3	DNA7AD5	DNA7AD6	DNA15AD2	DNA15AD3	DNA15AD5	DNA15AD6	DNA23AD2	DNA23AD3	DNA23AD5	DNA23AD6
DNA8AD2	DNA8AD3	DNA8AD5	DNA8AD6	DNA16AD2	DNA16AD3	DNA16AD5	DNA16AD6	DNA24AD2	DNA24AD3	DNA24AD5	DNA24AD6

**NotethatonlyleftborderisusedintheentireplateIfanalternatesetupisused,remembertomodifythecocktailforeachreactionviatheTAILRecipesetupsheet.TherecipecanbemanipulatedtoaccommaodateanynumberofADprimersandindividualDNAsamples.Ifthecocktailvolumeisgreaterthan1.5mL,twotubeswillbeneededtopreparethecocktail;divideeachcomponents""valuebytwoanduse2tubes.