Oligodesign

• The5"end(thehomologyarm)-choose42ormore(weusuallychooseabout50)ntsforthehomologyarmsfromthetargetDNAsequencesimplyaccordingtowhereyouwanttoinsertthePCRfragment.(Forefficientoligonucleotidesynthesis,theEMBLoligoserviceprefersthatthe5"mostnucleotideisaTorC.ConsequentlymostofouroligosstartwithaTorChoweverwedonotthinkthisisimportantforETcloningefficiencies).
• The3"end(thePCRprimer)-chooseabout18-30ntforthePCRprimerregionasyouwouldforanyotherPCR.UseoftheOligo4softwareprogramcanhelp,especiallyforshorterprimers.
• Betweenthesetworegions,youcanstickwhateveryoulike-withinthelimitsofoligosynthesis.WeroutinelyputloxPorFRTs(each34bps)here,forsubsequentrecombinationbyCreorFLPrecombinasesrespectively.

PCRreactionandrecipientplasmidDNApreparation

• Theoligopowder(40nmolwithoutFPLCpurification)wasdissolvedin500ulofdH2O.Extractoligosolutionwithphenol/chloroformasfollows:100uloligosolution12ul3MNaAc(pH7.5)120ulphenol:CHCl3Vortexfor30",spinfor2-3min,add360ulethanol,placein-80oCfreezerfor5-10min,spinfor5min,washoncewith70%ethanol,dryundervacuumanddissolvein100ulofdH2O.
• PCRreaction33uldH2O5ul10xPCRreactionbuffer5ul2.5mMdNTP(10x)1.5ulupperoligo1.5ulloweroligo2ultemplat0.5ulTaqpolymerase(5U/ul)Annealingtemperatureisimportant,usually60oC-62oCisoptimal.
• PurifythePCRproductsbyQiagencolumnandelutewith2x50uldH2O.
• Add10xDpn1bufferand2ulDpn1(NEB)anddigest(toeliminatetemplateDNA)for1hour.
• ExtractwithPhenol:CHCl3once,precipitatewithethanolasaboveandredissolvein5uldH2O(from50uloriginalPCRproducts,about0.3ug/ul)
• Ifcompetentcellsharbourrecipientplasmid,0.3ugofPCRproductwillbeusedfortransformation.
• Forco-transformation,extractrecipientDNAoncewithphenol:CHCl3asaboveanddissolveindH2Oat1ug/ul.MixPCRproductswithrecipientplasmidDNA(0.20-0.3pmolPCR+0.10-0.2pmolvectorperul).1ulofmixturewillbeusedfortransformation.

Preparationofcompetentbacterialcells

• TransformyourhostwiththeETexpressionplasmidyouareusingbystandardproceduresandplate.
• Picksinglecolonyandgrowin5mlLBmediumovernight.
• Transfer0.7mlinto70mlofLBmedium(withoutglucose!)andgrowthemat37oCwithshaking.
• Prepare10%glycerolwithdH2O,andcooldownoniceforatleast3hoursbeforeusing.
• WhenthecellsreachOD600=0.1-0,15,add0.7ml10%L-arABInosetoinduceETproteinexpression.
• Afterafurther45-60minutes,thecellsshouldbeatOD600of0.3-0.4.Harvest.
• makesurethecentrifugeandSA600orSS34(Sorvall)rotorisverycoldbycentrifugingfor10min,-5oCat4,000rpm.
• Spin35mlscellsfor10minat7,000rpmat-5oC.Puttheother35mlsonice.
• Pourawaythesupernatant,addthesecond35mlsandrespin.
• Pourawaysupernatant,puttubeonice,resUSPendcellsin5mlicecold10%glycerolwithanicecold5mlPipette.Addafurther25mlsandcentrigue.
• Repeattheabovesteptwice.
• PourawaysupernatantandimmediatelydrythetubeoutwithKleenextissuetakningcarenottotouchthepellet.
• Resuspendthecellsintheremainingliquid(youshouldhavealittlemorethan100ulfinalresuspendedvolume).
• Transfer50ulofcellsintoeachpre-cooledEppendorftubeandfreezeinliquidN2oruseimmediately.AllthestepsshouldbedoneascoldaspossIBLe,alwaysonice!Theglasspipettesshouldbecooledonceortwicebypipettingcold10%glycerolupanddownbeforepipettingcells.

Transformation

• (Ifcuvetteswerereused)Washcuvettesatleast10timeswithdH2O,precoolthemoniceforleast5min.
• Thawcompetentcellsoniceandadd1ulofPCRproduct(fortransformation)or1ulmixtureofPCRplusDNAforco-transformation.
• Electroporatethecellsat2.3kV(Bio-RadGenePulser),25uFwithPulsecontrollersetto200ohms)
• Add1mlofLBmediumandtransferbackintotheeppendorftube.
• Incubateat37oCfor1to1.5hourswithshaking.
• Plate100ulonsuitableantibiotocplates,spintherest,resuspendin100ulandplatethat..

UsingCreorFlptransientplasmids

705-Creand705-FlpplasmidsarebasedonthepSC101temperaturesensitiveorigin.Thisoriginmaintainsalowcopynumberandreplicatesat30oC.Theseplasmidswillbelostfromcellswhentheyareincubatedattemperaturesabove37oC.Inaddition,CreandFlpareexpressedfromthelamBDaPRpromoter,whichexpressesweaklyat30oCandstronglyat37oC.Thus705-Creand705-Flpcanbeusedtogiveatransientburstofexpressionafterwhichtheywillbeeliminatedsotherecombinedproductcanbeisolateduncontaminatedbytheirpresence.
• TransformtheE.colihostcontainingtheplasmidtobesitespecificallyrecombinedwith705-Flpor705-Cre.(Themethodoftransformationisnotcritical,weuseRbCl2competentcellsbecauseitissimplerthanpreparingelectrocompetentcells).
• Incubateat30oCfor1.5hrwithshakingaftertransformation.
• PlateonL.B.platescontaining25ug/mlofchloramphenicol(Cm).
• Incubateat30oCfor2days.
• Picksinglecoloniesandgrowamini-prepinL.B.mediumovernightat37-38oC(not35oC!).
• StreakontoL.B.agarplatewithoutchloramphenicol,andcultureat37oC.(Duringincubationat37oC,FlporCrewillbeexpressedandrecombineFRTorloxPsites.Atthesametime,the705plasmidwillbelost.)
• PicksinglecoloniesandstreakonCmplatestocheckifthe705plasmidislost.

UsingCreorFlpexpressingE.coli

294-Creand294-Flpweregeneratedbyintegrating705-Creand705-FlpintothelacZlocusofE.colistrain,MM294(Buchholzetal,NucleicAcidsRes.24,3118-9(1996).
• TransformtheplasmidcarryingloxPorFRTsitesinto294-Creor294-Flpcompetentcells.
• Incubateat37oCforonehour.
• Plateontheselectionplatesandincubateat37oCovernight.
• Pickcoloniesfromtheplatesandinoculate5mlLBmediumwithappropriateantibiotics.
• Incubateat30oCovernight.(Thetemperatureshouldbe30oCsothatCreorFlpexpressionisreduced.WeoftenobservethattheyieldofplasmidDNAfromcellsstronglyexpressingCreorFlpcanbeverylow.)
• Checktheplasmidbymini-preptoseetheloxPorFRTsiteslostornot.

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