HighEfficencyYeastPlasmidRescue

1. Growyeastovernight.
2. Spindown1.5mlofcultureinmicrofugetubes.
3. Decant
4. Add250µlofQiagenbufferP1toeachtube
5. Addabout250µlofglassbeads
6. Beatorvortexonhighfor3-5minutes
7. Add250µlofbufferP2andgentlymixwell(Donotletlysisreactioncontinueformorethanfiveminutes)
8. Add350µlofbufferN3andmiximmediately,butgently
9. Centrifugefor10minutesonfullblast
10. ApplysupernatantstoQIAprepspincolumn
11. Centrifugecolumnfor45secondsonfullblast.Discardflow-though
12. Washcolumnwith750µlofBufferPEandcentrifuge45seconds
13. Discardflowthroughandspinforanadditional45seconds
14. Placecolumninacleantubeandadd50µlofbufferEB
15. Letsitfor1min.thenspinfor1min.
16. Forefficenttransformationintocoliuse1-2µlofthisprepperaliquotofE.coliusingthe90secheatshockprotocol.(NOTE:usingupto20µlwillincreaseyieldoftransformants)

ColonyPlasmidRescue

ProtocoladaptedbyJuliaSidorovaandLindaBreedenfromHoffmanandWinston,Gene,1987,Vol.57:267-272.

1. Startwithafreshplateofyeast,withlargecoloniesgrownfor3-4days.
2. PrepareEppendorfswith20µlaqueousbuffer(2%TritonX-100,1%SDS,100mMNaCl,10mMTris-HClpH8.0,1mMEDTApH8.0).
3. PickcoloniesfromplatewithaPipettetipandresUSPendintheaqueousbuffer.Note:LablorerecommendsNOTusingatoothpicktopickyourcolony.Thereissomethingevilintoothpicksthatinhibitsbacterialtransformations.
4. Add10µlphenoland10µlchloroform(or20µlphenol:chloroform)toeacheppendorftube.
5. Add1/3eppendorfcap(0.65mltube)ofglassbeads.
6. Vortex5min.atspeedof4-5inmultiheadvortexeratroomtemperature.
7. Spinfor5min.atmaximumspeedinamicrocentrifuge.
8. ThawtransformationcompetentE.colicellsonice.
9. Intesttubes(formicrofugetubesseebelow;otherwiseuse14-mlsnap-captube)oniceadd:a)1µlaqueousphase(ifyouexperiencelowyield,tryusingmore)b)120µlcompetentcells
10. Mixandincubateonicefor30min.
11. Heatshockfor90secat42degC,thenimmediatelyadd2mlofNZYbrothtoeachtesttube.(LBwillalsowork,thoughrichermediaisusuallybetter)
12. Shakegentlyat37degCfor1hr.(Ifusingmicrofugetubes,rotateat37°Cinrollerdrum)
13. Pelletcellsfor5min.at3krpmintabletopcentrifuge.
14. Pouroffthesupernatant,resuspendthepelletintheresidualliquid,andplatetheentiresuspensiononselectivemedium.

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