Plasmid DNA minipreps相关交流-蚂蚁淘在线

DNAminipreps

TherearemanymethodsforDNApreparationwithoutusingkits.Belowareafewminipreps.Theseprotocolsmaybescaleuptomidiormaxiprepifnecessary.

1)AmmoniumAcetateMethod

1)SpindowncellandresUSPendin0.2mlSolution1.2)Lysecellbyadding0.4mlsolution2.Leavefor5min.onice.3)Add0.3ml7.5Mamm.acetate,spintopelletcelldebrisafter5minutesonice.4)Add0.6volisopropanol(0.54ml)tosupernatant,incubate10minutesandspin10-15minutes.5)Dissolvepelletin0.1ml2Mamm.acetate.Spinafter5min.onicetopelletproteins.6)Addequalvolumeofisopropanoltosupernatant,andspinfor10minutestoprecipitateDNA.7)Add50ulofRNasesolutiontodissolveDNA.Incubateat37°Cfor10minutes.8)PrecipitateDNAbyadding1/2vol.of7.5Mamm.acetateand3vol.isopropanol.Washpelletwith80%ethanol.9)Air-dryandresuspendDNAinTEbuffer.

2)Alternativeammoniumacetatemethod

Thisisashorterversionoftheabove.IftheDNAisnotcleanenough.addaphenol/chloroformstepattheend.

Pelletandresuspend1.5mlofo/nculturein200µlofbufferA.
Add400ulofbufferB,mixbyinversion,incubateonicefor5min.
Add300ulof7.5Mamm.acetate,mixandincubateonicefor10min.
Spinatfullspeed10min.
Transfersupernatantintonewtubewith500#181;lisopropanolandmixed.
Spin10-15min.
Discardsupernatantandwashpelletwith1mlof100%EtOH
drypelletsandresuspendin50µlH2OorTE.

BufferA:50mMTrisHClpH8.010mMEDTA100ug/mlRNaseA

BufferB:200mMNaOH1%SDS

7.5MNH4Ac(57.75g-madeupto100mlinwater)-preparedfreshmonthlyasammoniumacetatedecomposes.Note:1)TheoldestplasmidisolationmethodisbyClewellandHelinski(1969,Proc.Natl.Acad.Sci.USA,62,1159-1166)whichisaversionoftheTritonlysis,andalsoagentlemethodforpreparationoflargeDNA,butthechromosomalclotcanonlybepelletedathighspeeds.2)EndAstrains(e.g.DH5-alpha)issaidtobeunsuitableforboilingpreps(amyth?).3)Overnightculturecansometimesproducecatenae(interconnectedsupercoiledcircles),especiallywithveryhighcopynumberplasmidslikebluescript.IfcatenaeisaproblemorifyouwishtogethighestproportionofonlyformI-DNA,harvestcellatOD=1(1.5maximum).4)TogetonlysupercoiledDNA,purifyyourDNAoveraCsClgrADIentinthepresenceofEtBr.YoursupercoiledDNAformsabandwellseparatedfromrelaxedorlinearDNA.Twoconsecutivegradientsarebetterthanone.5)LysisbuffercontainingNaOHshouldbepreparedfresh-NaOHreactswithCO2fromair.7)TheLiClmethodcanalsobeused.TheRNase,however,isnotreallynecessaryforthisprep,asmostoftheRNAgoesintopelletbutsomelowMWRNAmayremain.6)AlkalinelysismethodscandenatureplasmidDNAwhichmakesomeDNAhardtodigest.Thereforeavoidleavingthesolutioninthealkalinestatefortoolongatroomtemperature.Alternativemethodsoflysiscanbeusedifitisaproblem(e.g.triton,boiling).8)OtherminiprepmethodsareavailableatMarkStrom"sprotocolssite.Seealso"Wicked-Quick"Mini-prep,MerlinMiniprepsandothers.9)References:Zhouetal.(Biotechniques,8(2)172-173[1990])Zinder&Boeckeboilinglysismethod-Gene19(1982)1-10Holmes&Quiglyboilingminiprep-AnalBiochem1981114(1):193-7BioTechniques,1997,vol23,No3,pp424-427.