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.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
