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Plasmid DNA minipreps
蚂蚁淘
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TherearemanymethodsforDNApreparationwithoutusingkits.Belowareafewminipreps.Theseprotocolsmaybescaleuptomidiormaxiprepifnecessary. 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.
================ 蚂蚁淘在线 ================ 免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容 版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。DNAminipreps 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.Solution1 Solution2 TEbuffer RNasesolution 50mMGlucose 1ml10%SDS 10mMTrisHClpH8 50ulof10mg/mlRNase 10mMEDTA 0.2ml10MNaOH 1mMEDTA 5mlTE 25mlTrisHClpH8.0 8.8mlwater - - 2)Alternativeammoniumacetatemethod
Thisisashorterversionoftheabove.IftheDNAisnotcleanenough.addaphenol/chloroformstepattheend.
BufferA:50mMTrisHClpH8.010mMEDTA100ug/mlRNaseA
