DNA Plasmid Miniprep Protocol
DNAPlasmidMiniprepProtocol 1.Picksinglecolonyandinoculate5mlofLBbrothcontaining200g/lampicillinor1mg/5ml.Optional:Usea15mlconicaltubewithaloosenedcapandapieceoftapetoholditinplace.Shakeat250RPM37oCovernight. 2.Centrifuge1.5mLcellsin1.5mLEppendorftubeattopspeedfor1minute.Aspiratesupernatant. 3.ResUSPendcellpelletin100ulofGTEbuffer(50mMGlucose,25mMTris-Cl,10mMEDTA,pH8).Vortexgentlyifnecessary. 4.Add200ulofNaOH/SDSlysissolution(0.2MNaOH,1%SDS).Inverttube6-8times. 5.IMMEDIATELYadd150ulof5Mpotassiumacetatesolution(pH4.8).ThissolutionneutralizesNaOHinthepreviouslysisstepwhileprecipitatingthegenomicDNAandSDSinaninsolublewhite,rubberyprecipitate.Spinattopspeed1min. 6.Transfersupernatanttonewtube,beingcarefulnottopickupanywhiteflakes.Precipitatethenucleicacidswith0.5mLofisopropanolonicefor10minutesandcentrifugeattopspeedfor1minute. 7.Aspirateoffalltheisopropanolsupernatant.Dissolvethepelletin0.4mlofTEbuffer(10mMTris-Cl,1mMEDTA,pH7.5).Add10ulofRNAseAsolution(20mg/mlstockstoredat-20°C),vortexandincubateat37°Cfor20to30minutestodigestremainingRNA. 8.ExtractproteinsfromtheplasmidDNAusingPCIA(phenol/chloroform/isoamylalcohol)byaddingabout0.3ml.Vortexvigorouslyfor30seconds.Centrifugeatfullspeedfor5minutesatroomtemperature.NoteorganicPCIAlayerwillbeatthebottomofthetube. 9.RemoveupperaqueouslayercontainingtheplasmidDNAcarefullyavoidingthewhiteprecipitatedproteinlayerabovethePCIAlayer,transferringtoaclean1.5mlepindorftube. 10.Add100mlof7.5Mammoniumacetatesolutionand1mlofabsoluteethanoltoprecipitatetheplasmidDNAonicefor10minutes.Centrifugeatfullspeedfor5minutesatroomtemperature. 11.AspirateoffethanolsolutionandresuspendordissolveDNApelletin50ulofDNA.Dissolve5uLin995ulofwater,andspec(blankspectrophotometertowater).Theabsorbanceat260nmmultipliedbytenistheconcentrationoftheDNAinunitsofmg/mlfora1cmpathlengthcuvette(i.e.50mg/ml/OD260nm).
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