1)Starta5mlo/nculture2)Pelleto/ncultureinapurplescrewcaptube(thismaytakeseveralspins),anddumps/n.3)Add100µlofSTETtopelletandvortexbriefly.4)Addaboutacapfullof0.45mmglassbeads(makesuretherearenobeadsinthecapthreadsandtightenwelltopreventleakage).5)Vortexfor5min.(8ifusingmultiheadvortex)atRT6)Addanother100µlofSTET,andvortexbriefly.7)Placetubesin100^oCheatblockfor3min.(covertubeswithweight).Alternatively,useboilingwaterbathforthisstep.8)Coolbrieflyonice(1-2min),andthenuseabluetiptotransferasmuchliquidaspossIBLeto1.5mlEppendorftube(discardtheglassbeads).9)Centrifugeat4^oCfor10min.10)Transfer100µlofs/ntoafreshtube.11)Add500µlof7.5Mammoniumacetate(storedat4^oC)andmixbriefly.12)Incubateat-20^oCfor1hrorovernight.13)Centrifuge10min.at4^oC(shouldseeasmallwhitepellet).14)Aliquot200µloficecold100%ethanolintotofresheppendorftubes.Transfer100µlofs/ntothesetubes.15)MixwellandletstandatRTforabout5min.16)Centrifuge10min.atRT17)Discards/n,washpelletwith200µlof70%ethanol.18)Centrifuge5min.,removes/n,drypelletinspeedvac.19)Re-sUSPendpelletin20µlofTEorwater,use10µltotransformcompetentbacteria.

STETbuffer:

•8%sucrosein50mMTrispH8.0•5%TritonX-100•50mMEDTA•7.5AmmoniumAcetate:28.9g/50mls

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