Version 2â 03_2019
iDeal Library Preparation kit(incl. Index Primer Set 1)
USER GUIDE
Cat. No.âC05010020â(24ârxns)
Pleaseâreadâthisâmanualâcarefullyâbeforeâstartingâyourâexperiment
TheâiDealâLibraryâPreparationâKitâhasâbeenâvalidatedâonâIP-StarâCompactâAutomatedâSystem.âTwoâversionsâofâprotocolâ(manualâandâautomated)âareâdescribedâinâthisâmanual.
3
Contents
Kitâmaterialsâ 4
Requiredâmaterialsânotâprovidedâ 6
Manual processing 7
Automated processing 16
Relatedâproductsâ 28
4 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
Kit materialsTheâiDealâLibraryâPreparationâKitâcontainsâallâreagentsânecessaryâforâtheâpreparationâ ofâ 24â libraries.â Theâ kitâ containsâ alsoâ theâ indexesâ allowingâforâ multiplexingâ upâ toâ 12â samples.â Theâ setâ ofâ additionalâ 12â indexesâ isâavailableâ separatelyâ (Indexâ Primerâ Setâ 2â (iDealâ Libraryâ Preparationâ Kitâx24),âDiagenode,âCat.âNo.âC05010021)âallowingâforâmultiplexingâupâtoâ24âsamples.â
Tableâ1.âReagentsâsuppliedâwithâtheâiDealâLibraryâPreparationâKit.
Description Storage
iDealâLibraryâEndâRepair/dA-TailingâEnzymeâMixâ(green) -20°C
iDealâLibraryâEndâRepair/dA-TailingâBufferâ(green) -20°C
iDealâLibraryâLigationâMasterâMixâ(red) -20°C
iDealâLibraryâLigationâEnhancerâ(red) -20°C
iDealâLibraryâPCRâMasterâMixâ(blue) -20°C
iDealâLibraryâAdaptorâforâIlluminaâ(red) -20°C
iDealâLibraryâUracilâExcisionâReagentâ(red) -20°C
iDealâLibraryâUniversalâPCRâPrimerâforâIlluminaâ(blue) -20°C
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Tableâ2.âListâofâindexesâsuppliedâwithâtheâiDealâLibraryâPreparationâKit.
Product Index Primer SequenceExpected
index Primer Sequence Read
Quantity Storage
iDealâLibraryâIndexâ1â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATCGTGATGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ ATCACG 10â”l -20°C
iDealâLibraryâIndexâ2â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATACATCGGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ CGATGT 10â”l -20°C
iDealâLibraryâIndexâ3â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ TTAGGC 10â”l -20°C
iDealâLibraryâIndexâ4â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ TGACCA 10â”l -20°C
iDealâLibraryâIndexâ5â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATCACTGTGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ ACAGTG 10â”l -20°C
iDealâLibraryâIndexâ6â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATATTGGCGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GCCAAT 10â”l -20°C
iDealâLibraryâIndexâ7â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATGATCTGGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ CAGATC 10â”l -20°C
iDealâLibraryâIndexâ8â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTCAAGTGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ ACTTGA 10â”l -20°C
iDealâLibraryâIndexâ9â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATCTGATCGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GATCAG 10â”l -20°C
iDealâLibraryâIndexâ10â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATAAGCTAGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ TAGCTT 10â”l -20°C
iDealâLibraryâIndexâ11â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACâ
TGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GGCTAC 10â”l -20°C
iDealâLibraryâIndexâ12â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTACAAGGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ CTTGTA 10â”l -20°C
Whereâ-s-âindicatesâphosphorothioateâbond.
NOTE:â Ifâ fewerâ thanâ 12â indexesâ areâ usedâ inâ aâ laneâ forâ sequencing,â itâ isârecommendedâtoâuseâtheâfollowingâindexes:
â- Poolâofâ2âsamples:âIndexâ#6âandâ12â- Poolâofâ3âsamples:âIndexâ#4,â6âandâ12â- Poolâofâ6âsamples:âIndexâ#2,â4,â5,â6,â7âandâ12
6 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
Required materials not providedâąâ100%âEthanolâąâNuclease-freeâWaterâąâ10âmMâTris-HCl,âpHâ8.0âorâ0.1XâTEâąâDNAâLoBindâTubesâ(Eppendorfâ#022431021)âąâAMPureÂźâXPâBeadsâ(BeckmanâCoulter,âInc.â#A63881)âąâMagneticârackâ-DiaMagâ0.2âmlâ(Diagenode,âCat.âNo.âB04000001)âąâPCRâMachineâąâOptional:âIndexâPrimerâSetâ2â(iDealâLibraryâPreparationâKitâx24)â
(Diagenode,âCat.âNo.âC05010021)
Tableâ3.âListâofâindexesâincludedâinâtheâkitâIndexâPrimerâSetâ2â(iDealâLibraryâPreparationâKitâx24).
Product Index Primer SequenceExpected
index Primer Sequence Read
Quantity Storage
iDealâLibraryâIndexâ13â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTGTTGACTGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ AGTCAA 10â”l -20°C
iDealâLibraryâIndexâ14â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATACGGAACTGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ AGTTCC 10â”l -20°C
iDealâLibraryâIndexâ15â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTCTGACATGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ ATGTCA 10â”l -20°C
iDealâLibraryâIndexâ16â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATCGGGACGGGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ CCGTCC 10â”l -20°C
iDealâLibraryâIndexâ18â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATGTGCGGACGTâGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GTCCGC 10â”l -20°C
iDealâLibraryâIndexâ19â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATCGTTTCACGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3 GTGAAA 10â”l -20°C
iDealâLibraryâIndexâ20â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATAAGGCCACGTâGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GTGGCC 10â”l -20°C
iDealâLibraryâIndexâ21â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTCCGAAACGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GTTTCG 10â”l -20°C
iDealâLibraryâIndexâ22â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATTACGTACGGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ CGTACG 10â”l -20°C
iDealâLibraryâIndexâ23â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATATCCACTCGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ GAGTGG 10â”l -20°C
iDealâLibraryâIndexâ25â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATATATCAGTGTGAâ
CTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ ACTGAT 10â”l -20°C
iDealâLibraryâIndexâ27â
PrimerâforâIllumina5ÂŽ-CAAGCAGAAGACGGCATACGAGATAAAGGAATGTGâ
ACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3ÂŽ ATTCCT 10â”l -20°C
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MANUAL PROCESSING
8 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
STEP 1EndâPreparation
Starting Material
5ângââ1â”gâfragmentedâDNA.
1.1 Mixâtheâfollowingâcomponentsâinâaâsterileânuclease-freeâtube:
iDeal Library End Repair/dA-Tailing Enzyme Mix (green) 3â”l
iDeal Library End Repair/dA-Tailing Buffer (green) 6.5â”l
FragmentedâDNA 55.5â”l*
TOTAL volume 65 ”l
*IfâDNAâInputâisâlessâthanâ55.5â”l,âcompleteâwithâwaterâupâtoâ55.5â”l.
1.2 Mixâbyâpipettingâfollowedâbyâaâquickâspinâtoâcollectâallâliquidâfromâtheâsidesâofâtheâtube.
1.3 Placeâinâaâthermocycler,âwithâtheâheatedâlidâon,âandârunâtheâfollowingâprogram:
âąâ30âminutesâatâ20°Câąâ30âminutesâatâ65°CâąâHoldâatâ4°C
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LSTEP 2AdaptorâLigation
NOTE:âIfâDNAâinputâisâ<â100âng,âdiluteâtheâiDeal Library Preparation Adaptor for Illuminaâ(providedâatâ15â”M)â1:10âinâ10mMâTRIS-HClâwithâ10âmMâNaClâtoâaâfinalâconcentrationâofâ1.5â”M.âUseâimmediately.
2.1 AddâtheâfollowingâcomponentsâdirectlyâtoâtheâiDealâLibraryâEndâPrepâreactionâmixtureâandâmixâwell:
iDeal Library Ligation Master Mix (red) 15â”l
iDeal Library Adaptor for Illumina* (red) 2.5â”l
iDeal Library Ligation Enhancer (red) 1â”l
TOTAL volume 83.5 ”l
2.2 Incubateâatâ20°Câforâ15âminutesâinâaâthermalâcycler.
2.3 Addâ3 ”l of iDeal Library Uracil Excision Reagentâ(red)âtoâtheâligationâmixture.
2.4 Mixâwellâandâincubateâatâ37°Câforâ15âminutes.
NOTE:âIfâneededâsamplesâcanâbeâstoredâovernightâatâ-20°C.
10 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
OPTION A
Size Selection of adaptor-ligated DNA
Sizeâselectionâisâoptional.âIfâtheâstartingâmaterialâisâlessâthanâ50âng,âsizeâselectionâisânotârecommended.âIfâyouâareânotâperformingâsizeâselection,âproceedâ toâ theâ nextâ pageâ andâ performâ clean-upâ stepâ priorâ toâ PCRâamplification.â Theâ followingâ sizeâ selectionâ protocolâ isâ forâ librariesâ withâ200â bpâ insertsâ only.â Forâ librariesâ withâ differentâ sizeâ fragmentâ inserts,âreferâtoâTableâ4âforâtheâappropriateâvolumeâofâbeadsâtoâbeâadded.âTheâsizeâselectionâprotocolâisâbasedâonâaâstartingâvolumeâofâ100â”l.
Tableâ4:âRecommendedâconditionsâforâbeadâbasedâsizeâselection.
Approximate insert size 150 bp 200 bp 250 bp 300 - 400 bp 400 - 500 bp 500 - 700 bp
Total Library size (insert + adaptor) 270âbp 320âbp 400âbp 400â-â500âbp 500â-â600âbp 600â-â800âbp
Volume of ligation reaction 100â”l
Volume of beads to add
1st Bead Selection 65â”l 55â”l 45â”l 40â”l 35â”l 30â”l
2nd Bead Selection 25â”l 25â”l 25â”l 20â”l 15â”l 15â”l
NOTE:âIfâneedeâtheâlibrariesâcanâbeâstoredâatâ-20°C.
2.5 VortexâAMPure XP beadsâtoâresuspend.
2.6 Addâ13.5 ”l dH2Oâtoâtheâligationâreactionâforâaâ100â”lâtotalâvolume.
2.7 Addâ55 ”l of resuspended AMPure XP beadsâtoâtheâ100â”lâligationâreaction.âMixâwellâbyâpipettingâupâandâdownâatâleastâ10âtimes.
2.8 Incubateâforâ5âminutesâatâroomâtemperature.
2.9 Quicklyâ spinâ theâ tubeâ andâ placeâ theâ tubeâ onâ theâ DiaMag02â -âmagneticârackâ(Cat.âNo.âB04000001)âtoâseparateâtheâbeadsâfromâtheâsupernatant.âAfterâtheâsolutionâisâclearâ(aboutâ5âminutes),âcarefullyâtransferâtheâsupernatantâcontainingâyourâDNAâtoâaânewâtubeâ(Caution: do not discard the supernatant).âDiscardâtheâbeadsâthatâcontainâtheâunwantedâlargeâfragments.
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2.10 Addâ25 ”l resuspended AMPure XP beadsâtoâtheâsupernatant,âmixâwellâandâincubateâforâ5âminutesâatâroomâtemperature.
2.11 Quicklyâspinâtheâtubeâandâplaceâitâonâtheâmagnetic rackâtoâseparateâtheâbeadsâ fromâtheâsupernatant.âAfterâ theâsolutionâ isâclearâ (aboutâ5â minutes),â carefullyâ removeâ andâ discardâ theâ supernatantâ thatâcontainsâ unwantedâ DNA.â Beâ carefulâ notâ toâ disturbâ theâ beadsâ thatâcontainâtheâdesiredâDNAâtargetsâ(Caution: do not discard beads).
2.12 Addâ200 ”l of 80% freshly prepared ethanolâtoâtheâtubeâwhileâinâtheâmagneticârack.âIncubateâatâroomâtemperatureâforâ30âseconds,âandâthenâcarefullyâremoveâandâdiscardâtheâsupernatant.
2.13 RepeatâStepâ2.12âonceâforâaâtotalâofâtwoâwashes.
2.14 Airâtheâdryâbeadsâforâ5âminutesâwhileâtheâtubeâisâonâtheâmagnetic rackâwithâtheâlidâopen.
2.15 EluteâtheâDNAâtargetâfromâtheâbeadsâintoâ17 ”l of 10 mM Tris-HCl or 0.1 X Te, pH 8.0.âMixâwellâonâaâvortexâmixerâorâbyâpipettingâupâandâdown.âIncubateâforâatâleastâ2âminutesâatâroomâtemperature.âQuicklyâspinâtheâtubeâandâplaceâ itâonâaâmagneticâstand.âAfterâ theâsolutionâisâ clearâ (aboutâ 5â minutes),â transferâ 15â ”lâ toâ aâ newâ PCRâ tubeâ forâamplification.
NOTE:âBeâsureânotâtoâtransferâanyâbeads.âTraceâamountsâofâbeadâcarryâoverâmayâaffectâ theâoptimalâperformanceâofâ theâpolymeraseâusedâ inâ theâ iDealâLibraryâPCRâMasterâMixâinâtheâsubsequentâPCRâstep.
12 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
OPTION B
Alternatively, Clean-up of adaptor-ligated DNA without Size Selection
2.5 VortexâAMPure XP beadsâtoâresuspend.
2.6 Addâ86.5 ”l resuspended AMPure XP beadsâtoâtheâligationâreaction.âMixâwellâbyâpipettingâupâandâdownâatâleastâ10âtimes.
2.7 Incubateâforâ5âminutesâatâroomâtemperature.
2.8 QuicklyâspinâtheâtubeâandâplaceâitâonâtheâDiaMag02â-âmagneticârackâ(Cat.âNo.âB04000001)âtoâseparateâbeadsâfromâsupernatant.âAfterâtheâsolutionâisâclearâ(aboutâ5âminutes),âcarefullyâremoveâandâdiscardâtheâsupernatant.âBeâcarefulânotâtoâdisturbâtheâbeadsâthatâcontainâDNAâtargetsâ(Caution: do not discard beads).
2.9 Addâ200 ”l of 80% freshly prepared ethanolâtoâtheâtubeâwhileâinâtheâmagneticârack.âIncubateâatâroomâtemperatureâforâ30âseconds,âandâthenâcarefullyâremoveâandâdiscardâtheâsupernatant.
2.10 RepeatâStepâ2.9âonce,âforâaâtotalâofâtwoâwashes.
2.11 Airâtheâdryâbeadsâforâ5âminutesâwhileâtheâtubeâisâonâtheâmagneticâstandâwithâtheâlidâopen.
2.12 Eluteâ theâ DNAâ targetâ fromâ theâ beadsâ byâ addingâ 17 ”l of 10 mM Tris-HCl, pH 8.0 or 0.1X Te.
2.13 NOTE:âBeâsureânotâ toâ transferâanyâbeads.âTraceâamountsâofâbeadâcarryâoverâmayâaffectâ theâoptimalâperformanceâofâ theâpolymeraseâusedâ inâ theâ iDealâ libraryâ PCRâ Masterâ Mixâ inâ theâ subsequentâ PCRâstep.
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2.14 Mixâwellâbyâpipettingâupâandâdown,âorâonâaâvortexâmixer.âIncubateâforâatâleastâ2âminutesâatâroomâtemperature.
2.15 Quicklyâspinâtheâtubeâandâplaceâitâonâtheâmagneticâstand.
2.16 Afterâtheâsolutionâisâclearâ(aboutâ5âminutes),âtransferâ15 ”lâtoâaânewâPCRâtubeâforâamplification.
14 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
STEP 3PCRâamplification
3.1 Mixâtheâfollowingâcomponentsâinâsterileâstripâtubes:
AdaptorâLigatedâDNAâFragments 15â”l
iDeal Library PCR Master Mix (blue) 25â”l
iDeal Library Index Primer* (blue) 5â”l
iDeal Universal PCR Primer* (blue) 5â”l
TOTAL volume 50 ”l
*âTheseâprimersâ(IndexâPrimerâSetâ1)âareâincluded.âIndexâPrimerâSetâ2âmayâbeâpurchasedâseparatelyâ(Cat.âNo.âC05010021).
NOTE:âIfâneededâtheâsamplesâcanâbeâstoredâat-20°C.
PCR cycling conditions
3.2 Transferâtubesâtoâaâpre-programmedâthermalâcyclerâandâincubateâasâfollows.
Cycle Step Temp Time Cycles
InitialâDenaturation 98°C 30âseconds 1
Denaturation 98°C 10âseconds4âââ12*
Annealingâ-âExtension 65°C 75âseconds
FinalâExtension 65°C 5âminutes 1
Hold 4°C â
*Suggestion:â4âPCRâcyclesâforâ1â”gâDNAâinput,â7â-â8âcyclesâforâ50âng,âandâ12âforâ5ângâDNAâinput.âFurtherâoptimizationâofâPCRâcycleânumberâmayâbeârequired.
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LClean-up of PCR amplification
3.3 VortexâAMPure XP beadsâtoâresuspend.
3.4 Addâ45 ”l of resuspended AMPure XP beadsâtoâtheâPCRâreactionsâ(~50â”l).âMixâwellâbyâpipettingâupâandâdownâatâleastâ10âtimes.
3.5 Incubateâforâ5âminutesâatâroomâtemperature.
3.6 QuicklyâspinâtheâtubeâandâplaceâitâonâDiaMag02â-âmagneticârackâtoâseparateâbeadsâfromâsupernatant.âAfterâtheâsolutionâisâclearâ(aboutâ5âminutes),âcarefullyâremoveâandâdiscardâtheâsupernatant.âBeâcarefulânotâtoâdisturbâtheâbeadsâthatâcontainâDNAâtargetsâ(Caution: do not discard beads).
3.7 Addâ 200 ”l of 80% ethanolâ toâ theâ tubesâ whileâ inâ theâ DiaMag02â -âmagneticârack.âIncubateâatâroomâtemperatureâforâ30âseconds,âandâthenâcarefullyâremoveâandâdiscardâtheâsupernatant.
3.8 RepeatâStepâ3.7âonce.
3.9 Airâdryâtheâbeadsâforâ5âminutesâwhileâtheâtubesareâonâtheâmagneticâstandâwithâtheâlidâopen.
3.10 Eluteâ DNAâ targetâ fromâ beadsâ intoâ 33 ”l of 0.1X TE.â Mixâ wellâ byâpipettingâ upâ andâ downâ atâ leastâ 10â times.â Incubateâ forâ atâ leastâ 2âminutesâatâroomâtemperature.âQuicklyâspinâtheâtubeâandâplaceâitâonâanâappropriateâmagneticâstandâtoâseparateâbeadsâfromâsupernatant.âAfterâtheâsolutionâisâclearâ(aboutâ5âminutes),âcarefullyâtransferâ30â”lâsupernatantâtoâaânewâPCRâtube.âStoreâlibrariesâatââ20°C.
3.11 CheckâtheâsizeâdistributionâonâanâAgilentâBioanalyserâHighâSensitivityâDNAâChip.âTheâsampleâmayâneedâtoâbeâdilutedâbeforeâloading.âAâ5âfoldâdilutionâcanâbeâused.â
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AUTOMATED PROCESSING
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OLProtocol
Theâ âIdeal_Library_Preparationââ protocolâ onâ theâ iP-StarÂźâ Compactâ isâusingâ theâ standardâ iDealâ Libraryâ Preparationâ Kitâ fromâ Diagenode.â TheâiDealâLibraryâPreparationâkitâallowsâtheâpreparationâofâindexedâlibrariesâofâgenomicâorâChIPâDNA.
Itâprovidesâflexibilityâtoâprepareâ1âtoâ32âlibrariesâinâoneârunâstartingâwithâ5ângâofâDNA.âTheâwholeâprotocolâtakesâapproximatelyâ1h30.âItâallowsâyouâtoâprepareâupâtoâ96âlibrariesâperâdayâwithâ3âruns.âAtâtheâend,âyouârecoverâligatedâproductsâreadyâforâamplification.
18 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
IP-Star setup1.1 SwitchâONâtheâIP-Star Compact.
1.2 SelectââProtocolsââiconâandâthenâclickâonââLibrary prepâ.
1.3 UnderââLibrary prepâ,âselectââIdeal_Library_Preparationâ.
NOTE:âIfâyouâplanâtoârunâbetween:
â- 1âandâ8âsamples,âchoseââIdeal_Library_Preparation_08ââ- 9âandâ16âsamples,âchoseââIdeal_Library_Preparation_16ââ- 17âandâ24âsamples,âchoseââIdeal_Library_Preparation_24ââ- 25âandâ32âsamples,âchoseââIdeal_Library_Preparation_32â
1.4 Setupâtheâexactânumberâofâsamplesâthatâyouâwantâtoâprocess.
NOTE:âTheâLeft Peltier Blockâisânowâcoolingâdownâtoâ4°Câtoâkeepâtheâenzymesâandâreagentsâcold.
1.5 Setupâallâtheâplasticsâonâtheâplatformâaccordingâtoâtheâscreenâlayout.
1.6 FillâTIP Rack 1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âwithâtipsâaccordingâtoâtheâscreen.
1.7 FillâLeft and Right Peltier Blocksâwithâ200â”lâtubeâstripsâaccordingâtoâtheâscreen.
Left PeltierBlock
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TIP Rack 1 TIP Rack 2 Right PeltierBlock
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STEP 1 & 2EndâPreparationâandâAdaptorâLigation
NOTE:âAllowâtheâreagentsâfromâiDeal Library Preparation Kitâtoâcomeâatâ4°C.âWorkâonâiceâfromâthisâpoint.
2.1 Prepareâtheâfollowingâmixes.
âąâiDealâLibraryâEndâPrepâMix:
Number of samples 1 8 16 24 32
iDeal Library End Repair/dA-Tailing Enzyme Mix (green)
3â”l 24â”l 48â”l 72â”l 96â”l
iDeal Library End Repair/dA-Tailing Buffer (green)
6.5â”l 52â”l 104â”l 156â”l 208â”l
TOTAL 9.5â”l 76â”l 152â”l 204â”l 280â”l
NOTE:â55.5 ”l of DNAâwillâbeâaddedâlaterâforâeachâsample.
âąâiDealâLibraryâAdaptorâLigationâMix:
Number of samples 1 8 16 24 32
iDeal Library Ligation Master Mix (red) 15â”l 120â”l 240â”l 360â”l 480â”l
iDeal Library Ligation Enhancer (red) 1â”l 8â”l 16â”l 24â”l 32â”l
TOTAL 16â”l 128â”l 256â”l 238â”l 512â”l
2.2 FillâtheâstripsâofâtheâLeft Peltier Blockâaccordingâtoâtheâscreenâlayoutâwithâtheâfollowingâreagents:
âąâLigation Mixâââ16â”lâperâwellâąâiDeal Library Adaptor for Illuminaâ(red)âââ2.5â”lâperâwellâąâiDeal Library Uracil Excision Reagentâ(red)âââ3â”lâperâwell
20 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
2.3 Fillâ theâ stripsâ ofâ theâ Right Peltier Blockâ accordingâ toâ theâ screenâlayoutâwith:
âąâiDeal Library End Prep Mixâ-â9.5â”lâperâwellâąâDNA sampleâââ55.5â”lâperâwell
2.4 CloseâtheâdoorâandâRun.
Well 9-12⹠Uracil Excision Reagent 3 ”l
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A B C D E F G H
Well 1-4Adaptor Ligation Mix 16 ”l⹠Ligation Master Mix 15 ”l
⹠Ligation Enhancer 1 ”l
Well 5-8⹠Adaptor for Illumina 2.5 ”l
PCR tubeLeft Peltier
Well 1-4iDeal Library End Prep 65 ”l⹠Fragmented DNA 55.5 ”l
⹠End Repair/dA-Tailing Enzyme Mix 3 ”l
⹠End Repair/dA-Tailing Buffer 6.5 ”l
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A B C D E F G H
Samples 25-32Samples 17-24Samples 9-16Samples 1-8
PCR tubeRight Peltier
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Size Selection of adaptor-ligated DNA
Sizeâselectionâisâoptional.âIfâtheâstartingâmaterialâisâlessâthanâ50âng,âsizeâselectionâisânotârecommended.âIfâyouâareânotâperformingâsizeâselection,âproceedâtoâtheâclean-upâstepâpriorâtoâPCRâamplification.
NOTE:âUseâtheâIP-StarâandâroomâtemperatureâAMPureâXPâbeadsâforâtheâsizeâselection.
2.5 ââSwitchâonâtheâIP-StarâandâselectââProtocolsââiconâandâthenââLibrary prepââcategory.
2.6 ââSelectââAMPure_XP_Size_Selection_08ââifâyouâplanâtoârunâbetweenâ1âandâ8âsamples,âorââAMPure_XP_Size_Selection_16ââifâyouâplanâtoârunâbetweenâ9âandâ16âsamples.
2.7 âSetupâ theâ exactâ numberâ ofâ samplesâ thatâ youâ wantâ toâ processâ byâpressingâtheâblackâbox.
NOTE:âTheâPeltierâBlockâ1âisânowâcoolingâdownâtoâ4°Câtoâkeepâyourâsamplesâcold.
2.8 Setupâallâtheâplasticsâonâtheâplatformâaccordingâtoâtheâscreenâlayout
âąâ âFillâTIP Rack 1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âwithâtipsâaccordingâtoâtheâscreen.
âąâ âFillâReagent Rack 1 & 2âwithâreagentâcontainersâaccordingâtoâtheâscreen.
âąâ âFillâ96 plate 1âwithâaâ96âwellâmicroplate.
âąâ âFillâPeltier Block 1âwithâ200â”lâtubeâstripsâaccordingâtoâtheâscreen.â
PeltierBlock 1
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96 plate 1
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96 plate 2
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TIP Rack 1 TIP Rack 2
ReagentRack 2
ReagentRack 1
PeltierBlock 2
22 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
2.9 Fillâtheârobotâwithâallâreagents.
âąâAddâ80 ”l of ChIP-seq grade waterâtoâeachâsampleâtoâhaveâaâfinalâvolumeâofâ100â”l.âPutâyourâsamplesâinâlaneâ1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âofâtheâPeltier Block 1.
âąâDistributeâtheâAMPureâXPâbeadsâaccordingâtoâtheârequiredâsizeâfollowingâtheârecommendationsâfromâtheâtableâ:
AMPure XP Final size
110â”l 250âbp
100â”l 280âbp
90â”l 325âbp
80â”l 400âbp
70â”l 500âbp
Weârecommendâtoâuseâtheâsizeâselectionâprotocolâforâtheâfinalâlibraryâsizeâofâ325âbpâ(insertâ+âadaptor).
NOTE:âResuspendâtheâbeadsâwithâpipettingâupâandâdownâseveralâtimesâbeforeâdispenseâthem.
âąâFillâtheâcontainerâofâtheâReagent Rack 1âwithâfreshlyâpreparedâ80%âEthanolâaccordingâtoâtheâscreen.
âąâFillâtheâcontainerâofâReagent Rack 2âwithâResuspensionâBufferâaccordingâtoâtheâscreen.
âąâCheckâtheâproperâinsertionâofâtheâracksâandâtheâconsumables.
2.10 CloseâtheâdoorâandâpressââRunââtoâstart.
2.11 âAfterâ theâ run,â recoverâ yourâ samplesâ onâ theâ upperâ rowâ ofâ theâ LeftâPeltierâBlock.âTheâfinalâvolumeâisâ20â”lâforâeachâsample.
2.12 âPressââOKââandââBackââuntilâtheâhomepageâappearsâonâtheâscreen.âPressââShutdownââandâwaitâuntilâtheâscreenâisâblackâbeforeâswitchingâoffâtheâIP-Star.
NOTE:âRemoveâallâtheâplasticsâfromâtheâplatform,âemptyâtheâwasteâshuttleâandâcleanâtheâinnerâsideâofâtheâIP-Starâwithâ70%âethanol.
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Alternatively, Clean-up of adaptor ligated DNA without size selection
NOTE:âUseâtheâIP-StarâandâroomâtemperatureâAMPureâXPâbeadsâforâtheâclean-up.
2.5 SelectââProtocolsââiconâandâthenââIPureââcategory.â
2.6 SelectââAMPure_XP_Purification_08ââifâyouâplanâtoârunâbetweenâ1âandâ8âsamples,âorââAMPure_XP_Purification_16ââifâyouâplanâtoârunâbetweenâ9âandâ16âsamples.
2.7 Setupâ theâ exactâ numberâ ofâ samplesâ thatâ youâ wantâ toâ processâ byâpressingâtheâblackâbox.
NOTE:âTheâPeltierâBlockâisânowâcoolingâdownâtoâ4°Câtoâkeepâyourâsamplesâcold.
2.8 Setupâallâtheâplasticsâonâtheâplatformâaccordingâtoâtheâscreenâlayout.
âąââFillâTIP Rack 1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âwithâtipsâaccordingâtoâtheâscreen.
âąââFillâReagent Rack 1 & 2 withâreagentâcontainersâaccordingâtoâtheâscreen.
âąââFillâ96 plate 1âwithâaâ96âwellâmicroplate.
âąââFillâPeltier Block 1 withâ200â”lâtubeâstripsâaccordingâtoâtheâscreen.
PeltierBlock 1
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101112
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101112
12
34
56
78
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1112
96 plate 1
A B C D E F G H
12
34
56
78
910
1112
96 plate 2
A B C D E F G H
TIP Rack 1 TIP Rack 2
ReagentRack 2
ReagentRack 1
PeltierBlock 2
24 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
2.9 Fillâtheârobotâwithâallâreagents
âąâPutâyourâsamplesâinâlaneâ1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âofâtheâPeltier Block 1.
âąâDistributeâ86.5 ”lâroomâtemperatureâAMPure XP beadsâinâeachâwellâofârowâ1â(andâ5âifâprocessingâmoreâthanâ8âsamples)âofâtheââ96-well Microplate.
NOTE:âResuspendâtheâbeadsâwithâpipettingâupâandâdownâseveralâtimesâbeforeâdispenseâthem.
âąâFillâtheâcontainerâofâtheâReagent Rack 1âwithâfreshlyâpreparedâ80%âEthanolâaccordingâtoâtheâscreen.
âąâFillâtheâcontainerâof Reagent Rack 2âwithâResuspensionâBufferâaccordingâtoâtheâscreen.
âąâCheckâtheâproperâinsertionâofâtheâracksâandâtheâconsumables.
2.10 CloseâtheâdoorâandâpressââRunââtoâstart.
2.11 Afterâtheârun,ârecoverâyourâsamplesâonâtheâupperârowâofâtheâPeltier Block 1.âTheâfinalâvolumeâisâ20â”lâperâeachâsample.
2.12 PressââOKââandââBackââuntilâtheâhomepageâappearsâonâtheâscreen.âPressââShutdownââandâwaitâuntilâtheâscreenâisâblackâbeforeâswitchingâoffâtheâIP-Star.
NOTE:â Removeâ allâ theâ plasticsâ fromâ theâ IP-Starâ platform,â emptyâ theâwasteâ shuttle,â andâ cleanâ theâ innerâ sideâ ofâ theâ IP-Starâ withâ 70%â ethanol.â
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STEP 3PCRâamplification
3.1 Mixâtheâfollowingâcomponentsâinâsterileâstripâtubes:
AdaptorâLigatedâDNAâFragments 15â”l
iDeal Library PCR Master Mix (blue) 25â”l
iDeal Library Index Primer* (blue) 5â”l
iDeal Universal PCR Primer* (blue) 5â”l
TOTAL volume 50 ”l
*â Theseâ primersâ (Indexâ Primerâ Setâ 1)â areâ included.â Indexâ Primerâ Setâ 2â mayâ beâpurchasedâseparatelyâ(Cat.âNo.âC05010021).
NOTE:âIfâneededâtheâsamplesâcanâbeâstoredâat-20°C.
PCR cycling conditions
3.2 Transferâtubesâtoâaâpre-programmedâthermalâcyclerâandâincubateâasâfollows.
Cycle Step Temp Time Cycles
InitialâDenaturation 98°C 30âseconds 1
Denaturation 98°C 10âseconds4âââ12*
Annealingâ-âExtension 65°C 75âseconds
FinalâExtension 65°C 5âminutes 1
Hold 4°C â
*Suggestion:â4âPCRâcyclesâforâ1â”gâDNAâinput,â7â-â8âcyclesâforâ50âng,âandâ12âforâ5ângâDNAâinput.âFurtherâoptimizationâofâPCRâcycleânumberâmayâbeârequired.
26 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
Clean-up of PCR amplification
NOTE:âUseâtheâIP-StarâandâroomâtemperatureâAMPureâXPâbeadsâforâtheâclean-up.
3.3 SelectââProtocolsââiconâandâthenââIPureââcategory.â
3.4 SelectââAMPure_XP_Purification_08ââifâyouâplanâtoârunâbetweenâ1âandâ8âsamples,âorââAMPure_XP_Purification_16ââifâyouâplanâtoârunâbetweenâ9âandâ16âsamples.
3.5 Setupâ theâ exactâ numberâ ofâ samplesâ thatâ youâ wantâ toâ processâ byâpressingâtheâblackâbox.
NOTE:âTheâPeltierâBlockâisânowâcoolingâdownâtoâ4°Câtoâkeepâyourâsamplesâcold.
3.6 Setupâallâtheâplasticsâonâtheâplatformâaccordingâtoâtheâscreenâlayout.
âąââFillâTIP Rack 1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âwithâtipsâaccordingâtoâtheâscreen.
âąââFillâReagent Rack 1 & 2 withâreagentâcontainersâaccordingâtoâtheâscreen.
âąââFillâ96 plate 1âwithâaâ96âwellâmicroplate.âąââFillâPeltier Block 1 withâ200â”lâtubeâstripsâaccordingâtoâtheâ
screen.
3.7 Fillâtheârobotâwithâallâreagents
âąâPutâyourâsamplesâinâlaneâ1â(andâ2âifâprocessingâmoreâthanâ8âsamples)âofâtheâPeltier Block 1.
âąâDistributeâ45 ”lâroomâtemperatureâAMPure XP beadsâinâeachâwellâofârowâ1â(andâ5âifâprocessingâmoreâthanâ8âsamples)âofâtheââ96-well Microplate.
NOTE:âResuspendâtheâbeadsâwithâpipettingâupâandâdownâseveralâtimesâbeforeâdispenseâthem.
âąâFillâtheâcontainerâofâtheâReagent Rack 1âwithâfreshlyâpreparedâ80%âEthanolâaccordingâtoâtheâscreen.
âąâFillâtheâcontainerâof Reagent Rack 2âwithâResuspensionâBufferâaccordingâtoâtheâscreen.
27
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OLâąâCheckâtheâproperâinsertionâofâtheâracksâandâtheâconsumables.
3.8 CloseâtheâdoorâandâpressââRunââtoâstart.
3.9 Afterâtheârun,ârecoverâyourâsamplesâonâtheâupperârowâofâtheâPeltier Block 1.âTheâfinalâvolumeâisâ20â”lâperâeachâsample.
3.10 PressââOKââandââBackââuntilâtheâhomepageâappearsâonâtheâscreen.âPressââShutdownââandâwaitâuntilâtheâscreenâisâblackâbeforeâswitchingâoffâtheâIP-Star.
NOTE:â Removeâ allâ theâ plasticsâ fromâ theâ IP-Starâ platform,â emptyâ theâ wasteâshuttle,âandâcleanâtheâinnerâsideâofâtheâIP-Starâwithâ70%âethanol.
28 IDEAL LIBRARY PREPARATION KITâ âMANUALâ
Related productsProduct Cat. No.
iDealâChIP-SeqâKitâforâHistones C01010051
AutoâiDealâChIP-seqâKitâforâHistones C01010171
BioruptorâPico B01060010
IP-StarâCompactâAutomatedâSystem B03000002
DiaMagâ0.2âmlâ-âmagneticârack B04000001
MagMeDIP-seqâkit C02010023
AutoâMagMeDIP-seqâkit C02010016
29
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