DNA Plasmid Miniprep Protocol

DNAPlasmidMiniprepProtocol

1.Picksinglecolonyandinoculate5mlofLBbrothcontaining200g/lampicillinor1mg/5ml.Optional:Usea15mlconicaltubewithaloosenedcapandapieceoftapetoholditinplace.Shakeat250RPM37^oCovernight.

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).