1、Select1stenzyme:Select2ndGOenzyme:EnzymeCat#TempSuppliedNEBufferSupplements%ActivityinNEBufferBSASAM1DoubleDigestRecommendation(s)forBamHI+Kpnl:Doubledigestisnotrecommended.Asequentialdigestisrequired,usingeachenzymeinitssuppliedNEBuffer.Note:*BamHIhasaHighFidelityversionBamHI-HF(R3136)KpnlhasaHighF

2、idelityversionKpnI-HF(R3142)DoubleDigestsDigestingaDNAsubstratewithtworestrictionendonucleasessimultaneously(doubledigestion)providereactionconditionsthatoptimizeenzymeactivityaswellasavoidstaractivityassociatesuppliedwithitsoptimalNEBuffertoensure100%activity.NEBuffercompositionsarelistedonIChartfo

3、rRestrictionEnzymesratesthepercentageactivityofeachrestrictionendonucleaseinPleaseuseNEBDoubleDigestFindertoperformadoubledigestcalculation.SuggestedNEBuffersforDoubleDigestionEnzymeAatIIAvrIIBamHIBglIIBsgIEagIEcoRIEcoRVHindIIIKpnIMseINcoINdeINNEBuffer443343EcoRI321434Aatll4-4seqseq4seqseq444444AvrI

4、I44-3243EcoRI221444BamHI3seq3-333EcoRI3seqseq333BglII3seq23-33EcoRI322233Bsgl44433-seqseq42seq444EagI3seq333seq-EcoRI3seqseq333EcoRIEcoRIseqEcoRIEcoRIEcoRIseqEcoRI-EcoRIseq1EcoRIEcoRIEcoRIEcoRV3423343EcoRI-22232HindIII242seq22seqseq2-2222KpnI141seq2seqseq122-111MseI4443243EcoRI221-44NcoI3443343EcoRI

5、3214-4NdeI4443343EcoRI22144-NheI242seq24seq1221224NotI3seq33333EcoRI322233PstI3433333EcoRI321333PvuI3seq23333EcoRI322333SacI141seq24seq1221414SacII444seq24seqEcoRI224444SalI3seq33333EcoRI3seqseq333SmaI444seqseq4seqseq44seq444SpeI444seq24seqEcoRI221444SphI2423243EcoRI221222XbaI4443243seq222444XhoI444

6、3343EcoRI321444XmaI444seq24seqseq4seq4444Note:EnzymesinDarkRedareavailableinHighFidelity(HF)Format.HFEnzymeshavebeener4whichmaysimplifyyourdoubledigest.ClickformoreHFinformation.SettingupaDoubleDigestionChooseanNEBufferthatresultsinthemostactivityforbothenzymes.IfstaractivityisausingoneofourHighFide

7、lity(HF)enzymes.IfBSAisrequiredforeitherenzyme,addittothedoubledigestreaction(BSAdoesnotinhenzyme).Setupreactionaccordingtorecommendedconditions.Thefinalconcentrationofglycercshouldbelessthan5%tominimizethepossibilityofstaractivity.Forexample,ina50plreamountofenzymeaddedshouldnotexceed5pl.Incubateat

8、recommendedtemperature.OvernightdoubledigestsshouldbeavoidepossibilityofstaractivityIftwodifferentincubationtemperaturesarenecessary,choosetheoptimalreasetupreactionaccordinglyAddthefirstenzymeandincubateatthedesiredtemperature,firstenzyme,addthesecondenzymeandincubateattherecommendedtemperature.Dep

9、endingonanenzymesactivityratinginanon-optimalNEBuffer,thenumberofunitsmaybeadjustedtocompensatefortheslowerrateofcleavage.SettingupaDoubleDigestionwithaUniqueBuffer(designated“U”)Ourbuffersystemhasbeenstreamlined,leavingthreeenzymesthathaveuniquebufferinthischartandtheRestrictionEnzymeActivityChart)

10、,SspI(samebuffercompositionasEc(mostcases,DpnIIrequiresasequentialdigest.SettingupaSequentialDigestionSetupareactionusingtherestrictionendonucleasethathasthelowestsaltconcentrati(recommendedbufferandincubatetocompletion.Adjustthesaltconcentrationofthereaction(usingasmallvolumeofaconcentratedsalappro

11、ximatethereactionconditionsofthesecondrestrictionendonuclease.Addthesecondenzymeandincubatetocompletethesecondreaction.Alternatively,aspincolumncanbeusedtoisolatetheDNApriortothesecondreaction.DoubleDigestFAQSpotlight:Q:IwouldliketodigestDNAwithEcoRIandXbaIatthesametime.TheDoubleDigestFindersequenti

12、aldigest,usingeachenzymeinitssuppliedNEBuffer.However,bothoftheseenzymactivityinNEBuffer2.Whyisadoubledigestnotrecommendedwiththeseenzymes?A:AlthoughEcoRIshows100%activityinNEBuffer2,italsoexhibitssignificantstaractivityinthinnon-specific,unintendedcleavage.ThisisalsoobservedwhenusingNEBuffer4.Forth

13、isreas(相繼的)digestisrecommendedHowever,researchhasshownthatEcoRIexhibitslesssNEBuffers1and3.SinceXbaIhas75%activityinNEBuffer3adoubledigestcouldbeperformprovidedthatthereactionsaresetupaccordingtotherecommendedreactionconditionsforavcInthiscase,EcoRIisavailableinHighFedelity(HF)Format.HFEnzymeshavebe

14、enengineersactivityandhave100%activityinNEBuffer4whichmaysimplifyyourdoubledigest.DoubleDigestFinderDigestinNEBuffer4at37C.CleavingaDNAsubstratewithtworestrictionenzymessimultaneously(doubledigestion)isacommontimesavingprocedure.UsethistooltoselectreactionconditionsamenabletoanytwoNEBrestrictionenzymes.Clickhereformoreinformationondoubledigestsandachartofdoubledigestsforsomecommonenzymecombinations.DigestinNEBuffer4at37C.DigestinNEBuffer4at37C.Select1stenzyme:Select2ndenzyme:GOEnzymeCat#