Diatomaceous Earthbased Midiprep
来自 : 蚂蚁淘
DiatomaceousEarth-basedMidi-prepNote:Thisprocedureisthemethodofchoiceforisolatingdoublestrandedplasmid-basedtemplatesfortheSequenaseDye-LabeledTerminatorSequencingReactions.A.Materials
Mediaandreagentsarelistedhereinthemaxi-prepsection.
B.Methods
- Pickonecolonyinto3mlof2xTYcontaingtheappropriateantibiotic(typicallyAmpat25ug/mlforpUC-basedplasmids)andincubateat37oCwithshakingfor8-9hours.
- Transferseedcultureto50mlmediaandincubateasabovefor11-14hours.
- Harvestcellsbytransferringintoa50mlsterileconicaltubeandspinningat3000rpminGPRtabletopcentrifugefor5min.Decantsupernatantandfreezecellpelletat-70oCforatleast1hour.
- ResUSPenddefrostedcellsin2mlGET/lysozyme(2mg/ml).Add4mlofNaOH/SDSandincubateonicefor5min.
- Add4mlof3MNaOAc,pH4.8,andincubateonicefor60min.
- Filterthesupernatantthroughcheeseclothintoanother50mlconicaltubeandspinat3000rpmintheGPRcentrifugefor20min.
- Decantsupernatantto50mlpolypropylenetubeandadd20mg/mlofDNase-freeRNaseA.Incubateat37oCfor30minutes.
- Add7ml(equalvolume)ofmatrix(20mg/mlinguanADIneHCldissolvedin10:1TE).Mixabout5min.Spin5minat3000rpminGPRtabletopcentrifuge.
- Washwithanequalvolumeofwashbuffer.Spin.Decantsupernatant.
- Washwithanequalvolumeofacetone.Spin.Decantsupernatant.
- Dryinvacuumoven(about15-20min).
- Add600ulof10:1TE,incubatefor10minat65oCwithintermittentmixing.
- Spinat8000for5min.DecantsupernatanttoEppendorftube.
- SpinagainandtransfertoEppendorftube(about250ul).
- Precipitatewith2.5volumesofethanol/acetate,rinsewith80%ethanol,anddryinaSpeed-vac.
- Resuspendin40ulof10:0.1TEandassayconcentrationusing2ulonanagarosegel.Theyieldshouldbe~1ug/mlofstartingcells.Use~5ugintheSequenaseDye-LabeledTerminatorSequencingReactions.
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