分子克隆操作流程英文版clone experimental protocol

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1、TuesdayPCR1)Preparea0.2mlcentrifugetubeforeachsample.2)AddreagentasTable1.Table1Reactionsystem(50μltotal)ComponentsVolumes(μl)Template(humangenomicsDNA)110×Taqbuffer5MgCl2410mMdNTP1Forwardprimer(10uM)1Reverseprimer(10uM)1TaqDNApolymerase1ddWater363)SetprogramasTable2onPRCinstrument.Table2Rea

2、ctionprocedureStepTemperature,oCtimeNumberofcyclesInitialdenaturation955min1Denaturation9530s30Annealing5430sExtension721minFinalelongation7210min1Finalhold4forever14)VerifythePCRproduct(~1000bp)byelectrophoresis.PlasmidPurification1)AssembleacolumnstackbyplacingaSpinColumnCP3intoacollection

3、tube.Add500μlBalanceLiquid(BL)toSpinColumnCP3andspinat12000rpmfor1minatroomtemperature.Discardtheliquidincollectiontube.AndreplaceCP3tocollectiontube.2)Transfer1mlbacterialcellsto1.5mlcentrifugetubeandspinat12000rpmfor1minatroomtemperature.Discardsupernatant.3)Resuspendthebacterialpelletin25

4、0μlofBufferP1.Thebacteriashouldberesuspendedcompletelybyvortexingorpipettingupanddownuntilnocellclumpsremain.4)Add250μlofBufferP2,mixthoroughlybyvigorouslyinvertingthesealedtube4–6times,andincubateatroomtemperaturefor2min.Donotvortex,asthiswillresultinshearingofgenomicDNA.5)Add250μlofBufferP

5、3,miximmediatelyandthoroughlybyvigorouslyinverting4–6times.Centrifugeat14000rpmfor10min.6)TransfersupernatanttoCP3promptly.Donotallowthepelleteddebristofallintothecolumn..Centrifugeat12000rpmfor1min.Discardtheliquidincollectiontube.AndreplacetheCP3tocollectiontube.7)Add600μlWashingBufferPWto

6、CP3andcentrifugeat12000rpmfor1min.Discardtheliquidincollectiontube.AndreplacetheCP3tocollectiontube.8)Repeatsteps7.9)Centrifugeat12000rpmfor2min,andair-dryfor10min.10)InserttheCP3intoa1.5mlcentrifugetube.Add35μlofNuclease-FreeWatertotheCP3andincubateatroomtemperaturefor2min.11)Centrifugeat12

7、000rpmfor2min.NanodropAnalysisForDNA1)Raisethesamplingarmandpipettethe2μlwaterontothelowermeasurementpedestal.2)SelecttheNucleicAcidapplicationfromthemainmenu.Ifthewavelengthverificationwindowappears,ensurethearmisdownandclickOK.3)SelecttheSampleTy