Inverse PCR for PACend sequencing

InversePCRforPAC-endsequencingfromBradBarbazuk

GoalistogeneratePCRfragmentsthatcontaintheendsofPACinsertsthatcanbesequenced.ForinversePCRwecutthePAConceinthevector(nearoutwardprimersite)andonceatunknownsiteininsert(andmanyotherplacesoutsidethesesites,thatdon"tmatter).Followingligationtogeneratesmallcircles,PCRisperformedwithoutwardfacingprimersattheendofthevectorthatwillamplifyaproductthatcontainstheendoftheinsert.Toincreasetheoddsofgettinganamplifiableproduct,weusetwodifferentenzymes(usedsingly!!)perpacend-NlaIIIandRsaIfortheSP6end,andNlaIIIandHinPI(orHhaI)fortheT7end.

PrimersPCYPAC2SP6LGCCGTCGACATTTAGGTGPCYPAC2SP6RGATCGAAACGGCAGATCG

PCYPAC2T7LCCTTGAGAGCCTTCAACCPCYOAC2T7RCGAGCTTGACATTGTAGGAC

Stagesinprotocol(1)RestrictiondigestofPACDNA(2)ligationofrestrictiondigests(3)PCRamplificationofligationproducts(4)SequencingofPCRproducts(5)Trimsequenceofprimerandvectorsequence.

(1)Restrictiondigests75-200ngPACDNAin20ulrestrictiondigest.

NlaIII___ulPACDNA2ulbuffer4(NEB)___ulH201ulNlaIII(10u)(orRsaI,orHinPI)20ultotal

note-usebuffer1forRsaI,buffer2forHinPIdigestions

Incubate2hrs37degrees.Youmaywanttorunaportion(5ul)ongeltoseethatdigestionhappened(nexttoundigestedPAClane).

HeatinactivateRestrictionenzyme.20min65degrees.(thiswillpreventredigestionduringligation).

(2)Ligationreactions..

2ulDigestion2ul10Xligationbuffer2ul10mMATP(ifnotinligationbuffer)13ulH201ul(400unitsNEB)ligase

Incubate2hrsRT.Youmaywanttorunaportionongelnexttodigeststoseeifevidenceofligation.

(3)PCRreactions(primersarestoredat10um)

SP6NlaIII	SP6RsaI	T7NlaIII	T7HinPI
5ulNlaIIIlig.rxn	5ulRsaIlig.rxn	5ulNlaIIIlig.rxn	5ulHinPIlig.rxn
1ulpcysp6L	1ulpcysp6L	1ulpcyT7L	1ulpcyT7L
1ulpcysp6R	1ulpcysp6R	1ulpcyt7R	1ulpcyT7R
18ulPCRmix	18ulPCRmix	18ulPCRmix	18ulPCRmix
0.1ulTAQ(0.5unit)	0.1ulTAQ	0.1ulTAQ	0.1ulTAQ

Now,weneedtoknowifanyoftheseamplifiedforseqeuncing.Take5ulofPCRreactiontogelswith100bpladdertogetaccuratesizes.

PCRreaction	MinimalExpectedSize	Problemsize(PACvectoralone)
NlaIII-SP6	72+ins	355
RsaI-Sp6	65+ins	79
NlaIII-T7	120+ins	125
HinPI-T7	114+ins	305

(4)SequencingChemistry

(a)ExoIII/SIAP(Shrimpalkalinephosphatase)treatmenttoremoveoligonucleotides5ulPCRproduct2ulexo/SAPmix(2ulexo,2ulSIAP,6ulH20formix)

15min,37degrees15min80degreestoinactivateenzymes

add7ulH20

(b)BigDyeTerminatorSequencingFollowlabprotocolforBigDyeSequencing.Weusea1/3volumereaction.

Instriptubes:

2.5ulBigDyeMix1ulTemplateDNA1ulprimer(oneoftheprimersusedforamplification)3.5ulH20

capSpindown10"togetallproductsmixedatbottom

Cyclesequencing-35cycles94C-15"45C-5"72C-2"

(c)PrecipitateDNAadd1ul3MNaOAc50ulcold100%ethanolspin30minutestoprecipitate(3500rpminplatecentrifuge)

washpellet2xwith100ul70%ethanoldrypellet,store-20tillreadytoload

(5)Trimvectorandprimersequencesfromreads.Usesequencebelowtotrimfromstartofdirectread.Ihaveincludedtheprimersequence,whichtogetherwiththefirstfewbaseswillnotshowuponyoursequence.Usereversecomplementofothermemberofpairtotrimendofread.InternalHinPandRsaIsitesarecapitalized.

pcytac2t7lccttgagagccttcaacccagtcagctccttccggtggGCGCggggCATG(NlaIIIsiteinvector)

pcypac2t7rcgagcttgacattgtaggactatattgctctAATaaatttgcggccgctaatacgactcactatagggagaGGATC(BamHIcloninginsertsite)

pcypac2sp6lgccgtcgacatttaggtgacactatagaGGATC(BamHIcloninginsertsite)

pcypac2sp6rgatcgaaacggcagatcgcaaaaacaGTACatacagaaggagaCATG(NlaIIIsite)