ThiswasprobablyderivedfromBirnboimandI"veuseditsince1984forisolationofrecombinantplasmidsfromE.coliforroutinescreening.Allsolutionsarelistedatthebottom."ul"=microliter

Fromovernightcultures(LB,BHI,otherE.colibroths):

1. Pour1.5mlinto1.5mlmicrofugetubes.
2. Centrifuge1mininmicrofugeandcarefullyaspirateoffthemedium.
3. Add100ulofSolutionIandresUSPendbyvortexing.
4. Add200ulofSolutionIIandmixcompletelybyinversion.Thecellsshouldlyseandturnsomewhatclearandviscous.
5. Letstand~3minandthenadd150ulofSolutionIII.Mixagainbyinversion--awhiteclotofDNA/protein/SDSshouldform.Incubateonice10-30min.
6. Centrifugefor5mininmicrofuge.
7. Pouroffthesupernatant(~400ul)intoafresh1.5mlmicrofugetube.Add1ml95%ethanolandmixwell.
8. Centrifugeinmicrofuge~15min.Carefullypouroffethanolandwashpelletwith0.5ml80%ethanol.Eitherdryinspeedvacordessicatewithawashof95%ethanolandairdry.
9. Resuspendpelletin100-300ulwaterorTE(volumedependentoncopynumberofplasmid).Readyforrestrictiondigestion,PCR,subcloning,etc.

"ul"=microliter

TheseprepscontainRNAsoifthisisaproblemastandardRNAsetreatmentwillsuffice.Furthercleanupcanbeaccomplishedwithphenol/chloroformextractionsbutforroutinescreeningandsubcloningIneverbother.Yourmileagemayvary.

References:

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