Description: ElectroLigase™combinesT4DNAligaseandanoptimized,ready-to-use2XreactionbuffercontainingaproprietaryligationenhancerandnoPEG.ThiscombinationisspecificallyformulatedtopromoterobustligationofalltypesofDNAends(blunt,sticky,TA).Itisdirectlycompat
IBL e,withoutdesaltingorpurification,withelectrocompetentcellsusedfortransformationbyelectroporation.Nothawingofthebufferisrequiredasitmaintainsaliquidstateduringstorageat-20°C*,therebysimplifyingreactionset-up.Byprovidinganoptimizedratioofenzymeandbuffercomponents,usersareabletorapidlyligatealltypesofDNAendsapplyingashortincubationtimeatroomtemperature.Ligationsforsubcloningcanbecarriedoutinsmallvolumeswithlowconcentrations,allowinguserstoconservepreciousDNAsamples.Thesereactionscanbe
Pipette ddirectly,withoutpurificationordilution,totransformmanystrainsofelectrocompetent
E.coli **.
*Freezersvaryintheiractualinternaltemperatures.Ourtestingdemonstratesthattheenzymeandbufferremainliquidat-20°C.
**ElectroLigaseisalsocompatiblewithchemicallycompetentstrainsof
E.coli .Performanceisgenerallyaround50%efficiency,whencomparedtotheBlunt/TALigaseMasterMix(
NEB #M0367).
Ligationreactionscontainingequalamounts(20ngvectorand3-foldmolarexcessofinsert)ofblunt-(A),T/A(B),orsticky-end(C)vector/insertpairsweresetupusingElectroLigaseandincubatedforthetimesshown.Afterheatinactivation,2μlofeachreactionwerewithdrawnanddirectlyusedtotransformNEB10-betaElectrocompetentE.coli (NEB#C3020).50μlaliquotsoftheoutgrowth(diluted,insomecases)wereplatedontoselectiveplatesandincubatedovernightat37°C.Colonieswerecounted,adjustedforplatingdilution,andgraphed. ProductSource Purifiedfroman
E.coli straincontainingarecombinantgeneencodingT4DNALigase.
ReactionVolumeDefinition 1XElectroLigaseReactionBufferwithDNAsubstratesand1μlElectroLigaseinan11μlreactionvolumeincubatedat25°Cfor30minutes.
Notes: Usage:Cells:Competentcellscanvarybyseverallogsintheircompetence.Perceivedligationefficiencydirectlycorrelateswiththecompetenceofthecellsusedfortransformation.Alwaystransformuncutvectorasacontrolforcomparisonpurposes.DNA:PurifiedDNAforligationscanbedissolvedindH2O(Milli-Q®waterorequivalentispreferable);TEorotherdilutebuffersalsoworkwell.Foroptimumligation,theamountofvectorDNAshouldbe20–100ngandtheinsertshouldbeaddedata3-foldmolarexcess.Forligationvolumesgreaterthan11μl,increasethevolumeofElectroLigaseReactionBufferaccordingly.Insert:vectorratiosbetween2and6areoptimalforsingleinsertions.Ratiosbelow2:1resultinlowerligationefficiency.Ratiosabove6:1promotemultipleinserts.IfyouareunsureofyourDNAconcentrations,performmultipleligationswithvaryingratios.TimeandTemperature:MostligationsperformedusingElectroLigasereachanendpointat60minutesorlesswhenperformedbetween4–37°C.Incubationbeyondthistimeprovidesnoadditionalbenefit.Ourrecommendationfora25°C(roomtemperature)incubationwaschosenafterevaluationofperformanceat4°C,16°C,25°C,and37°C.Mostconditionsreachedatleast50%performancewithin30minutes.
BIOLOG y:SomeDNAsequencesarenoteasytoclone.Sequencesthatformstructures,includinginvertedandtandemrepeats,areselectedagainstbyE.coli.SomerecombinantproteinsarenotwelltoleratedbyE.coliandcanresultinpoortransformationorsmallcolonies