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Overthepasttwomonths,wehavebeentestingvariousreactionconditionsfortheABIBig-DyeTerminatorswithdouble-strandedDNAtemplatesrangingfrompUCorpGEMwithoutanyinsertuptobacterialgenomesinthe2Mbpsize.Belowaretheresultsoftheseexperimentsandtheconclusionswehavereached.PLEASEBEADVISEDthatanydeviationfromtheABIprotocolsaspublishedinABI"sliteratureisnotsupportedbyABI.Ionlyamprovidingtheinformationbelowsoyouwillhaveanideaofwhatwehavedonebutandthedisclaimeris"usetheseprotocolsatyourownrisk".Ifyoulikethemandarehappyyoulearnedaboutthem,sendmesomee-mail.Iftheydon"tworkforyou,keepittoyourself...:-) Alsopleasebeawarethat1uloftheThermofidelaseenzymedoescostandthereforeTHERMOFIDELASEONLYSHOULDBEUSEDSPARINGLY!!Thus,ifyoudoneedtouseit,youcandilutetheThermofidelaseenzymewith5xreactionbufferatleast4-foldincaseyouneedit(seenotebelow).Finally,thereisnoreasontoeventhinkofusingThermofidelaseroutinelyforanysequencingotherthanbacterialgenomic.However,ifyouhavethe"gapfromhell"orthe"repeatfromhell"thenitmaybeworthatryalongwithincreasingtheextensiontemperatureto72degC,increasingtheDMSOconcentration,etc. Ifyouhaveanysuggestedchangestotheprotocolsbelow,pleaseletmeknowwhatexperimentsyouhavedoneandtheresults,sothatIcanimplementthem. Thanksandhappysequencing, --broe BelowarethesequencingconditionswehavetestedfortheABIBig-Dyeterminators(PE-ABI#4303150forthe1000reactionkit-Description:TF,KITBTDRR-1000)withvarioustemplates.Asusual,italwaysisimportanttoquantitatealltemplatesbyagarosegelelectrophoresisvssizeandconcentrationstandardsanddoafewtestswithdifferenttemplateconcentrationstodeterminetheoptimalconditionsforyourreactions. Althoughseveralconditionsaregivenbelow,theonesweroutinelyuseareunderlinedwith++++++"s.NoticealsothatyouSHOULDNOTREDUCEeithertheabsoluteamountofprimerortemplatewhenthereactionsarescaleddown. =============ForCosmids,BACs,PACs,Fosmids============= Note#1:Theresultsareimprovedusingamodificationoftheoriginalbacterialthermo-cyclingconditionsobtainedfromCherylHeinerratherthantheFScyclesequencingconditions,andbyincreasingthenumberofcyclesto100overnight.Typicallyweareusing18-22mersasprimerswherenomorethan65%identity(i.e.<14/20SmithWatermanIdentity)withalltheknownsequencesintheprojectdatabase,includingcloningandsequencingvectors.Withlongerprimers,e.g.30mers,theannealingtemperatureandextensiontemperatureeachshouldberaised5degCto55and65degCrespectivelytoreducenon-specificbinding. Note#2:Alloftheabovereactionsworkwith5to10%DMSO,withorwithoutThermofidelase.However,since1uloftheThermofidelasestockisrecommendedfora30or20ulreaction,theThermofidelasestockshouldbedilutedwith5xrxnbuffersuchthat:1ulofa1:2dilutionshouldbeusedinthe1/2reaction,and1ulofa1:4dilutionshouldbeusedinthe1/4reaction. Note#3:TheModifiedCosmid,BAC,PAC,FosmidThermo-cyclingConditions Note#4:Increasingthenumberofcyclesfrom45to60to80andeventuallyto100,almostlinearlyimprovedboththesignalandreadlength.Increasingtheannealingtemperaturefrom50to55degCandtheextensiontemperaturefrom60to65degCalsoimprovedthesignalandreadlengthbuttoalesserextentthanincreasingthenumberofcycles.Becauseoftheincreasednumberofcycles,theseincubationsonlyshouldbedoneovernight. Note#5:ThermoFidelaseimprovesthesignalandreadlengthwithbacterialgenomictemplatesforallprimerstested. Note#6:Althoughsomeprimersworkbetterthanothersandwehavenocluewhy,itisclearthatanyproposedprimershouldbescreenedagainstthealreadyknownsequencedataforthetargetcosmid,BAC,PAC,Fosmidclone,aswellassequencingvectorsequences.Thus,topreventtheprimerfrombindingtomultipleplacesonthetargetclone,theprimersequenceshouldbecomparedtoallthecontigsinthetargetclonedatabase.Onlythoseprimerswithahomologyoflessthan12/20SmithWatermanIdentityelsewhereonthetargetcloneshouldbeusedforcustomprimerdirectedsequencing.(i.e.Thus,toeliminatemultiplepriming,theprimer,whichhas100%sequenceidentitytothesiteyouwantittobind,shouldnothavemorethan60%homologyelsewhereinthetargetlargeinsertclone.) ThePrimOUcomputerprogramisavailablefromourinformaticsgroupthathasbeenmodifiedfromtheSWMedicalCenter"sPrimoprogram,toallowscreeningagainstmultiplecontigsinaphred/phrapdatabaseatvariousstringencies.Inaddition,typicallywechooseprimerswitheitheraGorContheir3"endandaroughlyevendistributionofeachofthe4basesoraslightlyhigherG/Ccontent(i.e.1-2moreG"sorC"sthanA"sorT"stoshifttheTmslightlyhigher) Note#7:Asoneincreasestheprimerlength,theannealingtemp.canbeincreasedsotoocantheextensiontemperature.Wehavetested20,25,30,35mersonbacterialgenomictemplatesaswellasincreasingtheextntempfrom60to65to72degCandtheannealingtemperaturefrom50to55,60,65,70degC. Ourconclusionisthat: *PE-ABI#4303150the1000reactionkit:Description:TF,KITBTDRR-1000**FideltySystemsInc.(301)527-8250BigDyeProtocolsandNotes-Cosmid,BAC,BAC,FosmidTemplates
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++++++++++++++Big-DyeProtocolsandNotes+++++++++++++++Reaction"size" BigDyeMix(ul) Primer(pmoles) Template(ngrams) H20tofinalvolumeof Num.ofCycles TIGR"s1.5xreaction 12ul 10-30 400-1000 30ul 25 ABI1xreaction 8ul 10-30 400-1000 20ul 45 OU1/2reaction 4ul 10-30 400-1000 10ul 60-99 OU1/4reaction 2ul 10-30 400-1000 5-7ul 99(seeNote#3)