A rapid alkaline extraction method for the isolation of plasmid
来自 : 蚂蚁淘
ThiswasprobablyderivedfromBirnboimandI"veuseditsince1984forisolationofrecombinantplasmidsfromE.coliforroutinescreening.Allsolutionsarelistedatthebottom."ul"=microliter
Fromovernightcultures(LB,BHI,otherE.colibroths):
- Pour1.5mlinto1.5mlmicrofugetubes.
- Centrifuge1mininmicrofugeandcarefullyaspirateoffthemedium.
- Add100ulofSolutionIandresUSPendbyvortexing.
- Add200ulofSolutionIIandmixcompletelybyinversion.Thecellsshouldlyseandturnsomewhatclearandviscous.
- Letstand~3minandthenadd150ulofSolutionIII.Mixagainbyinversion--awhiteclotofDNA/protein/SDSshouldform.Incubateonice10-30min.
- Centrifugefor5mininmicrofuge.
- Pouroffthesupernatant(~400ul)intoafresh1.5mlmicrofugetube.Add1ml95%ethanolandmixwell.
- Centrifugeinmicrofuge~15min.Carefullypouroffethanolandwashpelletwith0.5ml80%ethanol.Eitherdryinspeedvacordessicatewithawashof95%ethanolandairdry.
- Resuspendpelletin100-300ulwaterorTE(volumedependentoncopynumberofplasmid).Readyforrestrictiondigestion,PCR,subcloning,etc.
"ul"=microliter
TheseprepscontainRNAsoifthisisaproblemastandardRNAsetreatmentwillsuffice.Furthercleanupcanbeaccomplishedwithphenol/chloroformextractionsbutforroutinescreeningandsubcloningIneverbother.Yourmileagemayvary.
| SolutionI | SolutionII | SolutionIII |
|---|
| 5mMsucrose10mMEDTA25mMTris,pH8.0 | 0.2NNaOH1%(w/v)SDS | 3Msodiumacetate,pH4.8 |
References:
Birnboim,H.C.1983.ArapidalkalineextractionmethodfortheisolationofplasmidDNA.MethodsEnzymol.100,243-255[Abstract].
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