High Efficency Yeast Plasmid Rescue
来自 : 蚂蚁淘
HighEfficencyYeastPlasmidRescue
(byTreyPowers,FieldsLab)- Growyeastovernight.
- Spindown1.5mlofcultureinmicrofugetubes.
- Decant
- Add250µlofQiagenbufferP1toeachtube
- Addabout250µlofglassbeads
- Beatorvortexonhighfor3-5minutes
- Add250µlofbufferP2andgentlymixwell(Donotletlysisreactioncontinueformorethanfiveminutes)
- Add350µlofbufferN3andmiximmediately,butgently
- Centrifugefor10minutesonfullblast
- ApplysupernatantstoQIAprepspincolumn
- Centrifugecolumnfor45secondsonfullblast.Discardflow-though
- Washcolumnwith750µlofBufferPEandcentrifuge45seconds
- Discardflowthroughandspinforanadditional45seconds
- Placecolumninacleantubeandadd50µlofbufferEB
- Letsitfor1min.thenspinfor1min.
- Forefficenttransformationintocoliuse1-2µlofthisprepperaliquotofE.coliusingthe90secheatshockprotocol.(NOTE:usingupto20µlwillincreaseyieldoftransformants)
ColonyPlasmidRescue
ProtocoladaptedbyJuliaSidorovaandLindaBreedenfromHoffmanandWinston,Gene,1987,Vol.57:267-272.
- Startwithafreshplateofyeast,withlargecoloniesgrownfor3-4days.
- PrepareEppendorfswith20µlaqueousbuffer(2%TritonX-100,1%SDS,100mMNaCl,10mMTris-HClpH8.0,1mMEDTApH8.0).
- PickcoloniesfromplatewithaPipettetipandresUSPendintheaqueousbuffer.Note:LablorerecommendsNOTusingatoothpicktopickyourcolony.Thereissomethingevilintoothpicksthatinhibitsbacterialtransformations.
- Add10µlphenoland10µlchloroform(or20µlphenol:chloroform)toeacheppendorftube.
- Add1/3eppendorfcap(0.65mltube)ofglassbeads.
- Vortex5min.atspeedof4-5inmultiheadvortexeratroomtemperature.
- Spinfor5min.atmaximumspeedinamicrocentrifuge.
- ThawtransformationcompetentE.colicellsonice.
- Intesttubes(formicrofugetubesseebelow;otherwiseuse14-mlsnap-captube)oniceadd:a)1µlaqueousphase(ifyouexperiencelowyield,tryusingmore)b)120µlcompetentcells
- Mixandincubateonicefor30min.
- Heatshockfor90secat42degC,thenimmediatelyadd2mlofNZYbrothtoeachtesttube.(LBwillalsowork,thoughrichermediaisusuallybetter)
- Shakegentlyat37degCfor1hr.(Ifusingmicrofugetubes,rotateat37°Cinrollerdrum)
- Pelletcellsfor5min.at3krpmintabletopcentrifuge.
- Pouroffthesupernatant,resuspendthepelletintheresidualliquid,andplatetheentiresuspensiononselectivemedium.
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