DNAPlasmidMaxiprepProtocol

1.Picksinglecolonyandinoculate250mlofLBbrothcontaining100mg/lampicillinorappropriateantibiotic.Shakeat250RPMovernight.

2.CentrifugecellsinaSovallGSA(250ml)orSLA-3000(500ml)rotorat5k×gfor10minutes.

3.ResUSPendcellpelletin5mlofGTEbuffer(50mMGlucose,25mMTris-Cl,10mMEDTA,pH8)bypipettingupanddownwitha10mlPipette.Optional:addaspatulatipoflysozymepowder.Agoodsuspensionisconsistentwithoutclumpsofcellpellet.

4.Add10mlofNaOH/SDSlysissolution(0.2MNaOH,1%SDS).Useaspatulatostiranddissolvethecellsuntilthesolutionbecomesclear,yellow.Alternatively,youcanshakethebottlefor5seconds.

5.Quicklyadd7.5mlof5Mpotassiumacetatesolution(pH4.8).ThissolutionneutralizesNaOHinthepreviouslysisstepwhileprecipitatingthegenomicDNAandSDSinaninsolublewhite,rubberyprecipitate.Shakebottlethoroughly.Centrifugeat10k×gfor10minutes.

6.PourthesupernatantintoacleanSS-34tubethroughasmalltwo-plysquareofcheeseclothplacedinthecenterofafunnel.ThecheeseclothcatchesanyfragmentsofSDS/genomicDNApelletfloatingonthesurface.

7.Precipitatethenucleicacidsbyadding20mlofisopropanoltotheSS-34tubeandicefor10minutes.Centrifugeat10k×gfor10minutes.

8.Aspirateoffalltheisopropanolsupernatant.Dissolvethepelletin5mlofTEbuffer(10mMTris-Cl,1mMEDTA,pH7.5).Add5mlof5MLiClsolutiontoprecipitateRNA.Leaveonicefor10minutesandcentrifugeat10k×gfor10minutes.

9.PouroffthesupernatantcontainingplasmidDNAintoacleanSS-34tube.Addanequalvolumeofisopropanol(10ml)andprecipitatethenucleicacidsonicefor10minutes.Centrifugeat10k×gfor10minutes.

10.Aspirateoffalltheisopropanolsupernatant.Dissolvethepelletin1mlofTEbuffer.TransferTEsolutionintoa1.5mlepindorftube.Add15ulofRNAseAsolution(20mg/mlstockstoredat-20°C),vortexandincubateat37°Cfor20to30minutestodigestremainingRNA.

11.PrecipitatetheplasmidDNAwithPEGsolution(30%polyethyleneglycol,1.6MNaCl)byadding0.4mlandincubating1hr-overnightonice.ThisstepdiscriminatesverylargeplasmidDNAfromsmallnucleicacidfragmentsasonlythelargerplasmidDNAprecipitate.

12.SpinthePEGsolutioninthecentrifugeatfullspeedforoneminute.AspirateoffthesupernatantPEGbufferanddissolvethePEGpelletin0.4mlofTEbuffer.Ifitisdifficulttoresuspendwithpipetteman,letitsitatroomtemperaturefor10minutesandtryagain.

13.ExtractproteinsfromtheplasmidDNAusingPCIA(phenol/chloroform/isoamylalcohol)byaddingabout0.3ml.Vortexvigorouslyfor30seconds.Centrifugeatfullspeedfor5minutesatroomtemperature.NoteorganicPCIAlayerwillbeatthebottomofthetube.

14.RemoveupperaqueouslayercontainingtheplasmidDNAcarefullyavoidingthewhiteprecipitatedproteinlayerabovethePCIAlayer,transferringtoaclean1.5mlepindorftube.Ifyoucatchanyprecipitatewhenremovingaqueouslayer,addfreshPCIAandrepeat.

15.Add100ulof7.5Mammoniumacetatesolutionand1mlofabsoluteethanoltoprecipitatetheplasmidDNA,usuallyonicefor10minutes.Centrifugeatfullspeedfor5minutesatroomtemperature.

16.AspirateoffethanolsolutionandresuspendordissolveDNApelletin0.3to0.5mlofTEbuffer.ThisisthefinalstockofPEGpureplasmidDNAwhichissuitableforDNAsequencingandlongtermstorage.

17.MeasuretheconcentrationoftheplasmidDNAbydilutingstockintowaterat1:200or5mlofDNAper1mlofwater(blankspectrophotometertowater).Theabsorbanceat260nmmultipliedbytenistheconcentrationoftheDNAinunitsofmg/mlfora1cmpathlengthcuvette(i.e.50mg/ml/OD260nm).A250mlflaskshouldyieldabout0.5mgofDNA

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