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Preparation of Genomic DNA from Bacteria using Phase Lock GelTM

来自 : 蚂蚁淘

PreparationofGenomicDNAfromBacteria-usingPhaseLockGel^TM
(ModifiedfromExperimentalTechniquesinBacterialGenetics,JonesandBartlet,1990)
Materials:
TEbuffer
10%(w/v)sodiumdodecylsulfate(SDS)
20mg/mlproteinaseK
phenolchloroform(50:50)
isopropanol
70%ethanol
3MsodiumacetatepH5.2
PhaseLockGel^TM(5Prime,3Prime,Inc)
1.GrowE.colicultureovernightinrichbroth.Transfer1.5mltoamicrocentrifugetubeandspin2min.Decantthesupernatant.Repeatwithanother1.5mlofcells.DrainwellontoaKimwipe.
2.ResUSPendthepelletin467μlTEbufferbyrepeatedpipetting.Add30μlof10%SDSand3μlof20mg/mlproteinaseK,mix,andincubate1hrat37EC.
3.Addanequalvolumeofphenol/chloroformandmixwellbyinvertingthetubeuntilthephasesarecompletelymixed.CAUTION:PHENOLCAUSESSEVEREBURNS,WEARGLOVESGOGGLES,ANDLABCOATANDKEEPTUBESCAPPEDTIGHTLY.CarefullytransfertheDNA/phenolmixtureintoaPhaseLockGel^TMtube(green)andspin2min.
4.Transfertheupperaqueousphasetoanewtubeandaddanequalvolumeofphenol/chloroform.AgainmixwellandtransfertoanewPhaseLockGel^TMtubeandspin5min.Transfertheupperaqueousphasetoanewtube.
5.Add1/10volumeofsodiumacetate.Mix.
6.Add0.6volumesofisopropanolandmixgentlyuntiltheDNAprecipitates.
7.SpoolDNAontoaglassrod(orPasteurpipetwithaheat-sealedend).
8.WashDNAbydippingendofrodinto1mlof70%ethanolfor30sec.
9.ResuspendDNAin100-200μlTEbuffer.Completeresuspensionmaytakeseveraldays.
10.StoreDNAat4ECshortterm,-20or-80EClongterm
11.AfterDNAhasdissolved,determingtheconcentrationbymeasuringtheabsorbanceat260nm.

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发布于： 2021-09-16 阅读（157）

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	TEbuffer
	10%(w/v)sodiumdodecylsulfate(SDS)
	20mg/mlproteinaseK
	phenolchloroform(50:50)
	isopropanol
	70%ethanol
	3MsodiumacetatepH5.2
	PhaseLockGel^TM(5Prime,3Prime,Inc) 1.GrowE.colicultureovernightinrichbroth.Transfer1.5mltoamicrocentrifugetubeandspin2min.Decantthesupernatant.Repeatwithanother1.5mlofcells.DrainwellontoaKimwipe. 2.ResUSPendthepelletin467μlTEbufferbyrepeatedpipetting.Add30μlof10%SDSand3μlof20mg/mlproteinaseK,mix,andincubate1hrat37EC. 3.Addanequalvolumeofphenol/chloroformandmixwellbyinvertingthetubeuntilthephasesarecompletelymixed.CAUTION:PHENOLCAUSESSEVEREBURNS,WEARGLOVESGOGGLES,ANDLABCOATANDKEEPTUBESCAPPEDTIGHTLY.CarefullytransfertheDNA/phenolmixtureintoaPhaseLockGel^TMtube(green)andspin2min. 4.Transfertheupperaqueousphasetoanewtubeandaddanequalvolumeofphenol/chloroform.AgainmixwellandtransfertoanewPhaseLockGel^TMtubeandspin5min.Transfertheupperaqueousphasetoanewtube. 5.Add1/10volumeofsodiumacetate.Mix. 6.Add0.6volumesofisopropanolandmixgentlyuntiltheDNAprecipitates. 7.SpoolDNAontoaglassrod(orPasteurpipetwithaheat-sealedend). 8.WashDNAbydippingendofrodinto1mlof70%ethanolfor30sec. 9.ResuspendDNAin100-200μlTEbuffer.Completeresuspensionmaytakeseveraldays. 10.StoreDNAat4ECshortterm,-20or-80EClongterm 11.AfterDNAhasdissolved,determingtheconcentrationbymeasuringtheabsorbanceat260nm.