PreparationofElectrocompetentCellsandElectroporationofPlasmidDNA

1. Inoculate5-mlL-brothwithasinglecolonyofE.coli.Incubate5hourstoovernightat37°Conarollerorwithmoderateshaking.

2. InoculateavolumeofL-brothcontainedinanappropriatelysizedside-armflaskwithone-tenthvolumeoftheculture(i.e.5mlofcultureto500mlL-broth,2.5mlofculturein250mlL-broth).Growcellsat37°Cwithshaking(200-300rpm)toanOD600of0.5to0.6.

3. Chillthecellsinanice-waterbathfor10to15minutesandtransfertoapre-chilled250-mlcentrifugebottle.Dividethecultureifrequired.
   • Cellsshouldbekeptat2°Cforallsubsequentsteps.

   • Pelletthecellsbycentrifugationat4200rpm,2°Cfor20minutesusingaBeckmanGSArotor.

   • PouroffthesupernatantandresUSPendthecellsin.01volumeice-colddH2O(5mldH2Oforanoriginalculturevolumeof500ml;one-halfthisamounttoeachbottleiftheculturewasdividedinstep3.Divideeachsubsequentvolumeinhalfiftheculturewasdivided).Addavolumeofice-colddH2Oequaltotheoriginalculturevolumeandmixwell.Centrifugethecellsasinstep4.

   • Pouroffthesupernatantimmediatelyandresuspendthecellsinthesmallamountoffluidremaininginthebottle.
     • Thepelletmaybeveryloose.Exercisecareandpouroffthesupernatantimmediately.

     • Addanothervolumeofice-colddH2Oequaltotheoriginalculturevolumeandmixwell.Centrifugethecells,againasinstep4.Pouroffthesupernatantimmediatelyandresuspendthecellsintheremainingfluid.

     • Ifthecellsaretobeuseddirectlyforelectroporation:
       1. Placethecellsuspensioninapre-chilled,narrowbottomtubeofappropriatesizeandspinfor10minutesat4200rpm,2°C.

       2. Decantthesupernatant,andestimatethepellet"svolume.

       3. Addanequalvolumeofice-colddH2Otoresuspendthecells.

       4. Keepcellsoniceuntilelectroporationiscompleted.

       5. Ifthecellsaretobefrozenforlateruse:
          1. Placethecellsuspensioninanappropriately-sized,narrow-bottomtubethathasbeenpre-chilled.

          2. Addtothecellsanamountofice-cold10%glycerolequalto0.08oforiginalculturevolume(40mlforacultureoforiginally500ml)andmixwell.

          3. Spinfor10minutesat4200rpm,2°C.

          4. Decantthesupernatant,andestimatethepellet"svolume.

          5. Addanequalvolumeofice-cold10%glycerolandresuspendthecells(onice).

          6. Aliquot50-300µlofthecellstopre-chilledmicrocentrifugetubes.

          7. Freezethecellsbyincubationinadryice/ethanolbath.Storeat-80°C.

          8. Whenreadytouse,thawthecellsonice.

             ElectroporationoftheCells

             1. Settheelectroporationapparatusto2.5kV,25µF.Setthepulsecontrollerto200omega.

             2. Add1µlplasmidDNAtotubescontaining40µlfreshorthawedcellsonice.MixbyswirlingwithPipettetip.
                • ThevolumeofDNAaddedtothecellsshouldbekeptsmall.AddingDNAuptoone-tenththevolumeofcellscanreducetheefficiencyofelectroporation2-to3-fold.

                • TransfertheDNAandcellstoapre-chilledelectroporationcuvette(0.2cmelectrodegap)usinganarrowpipettetip.WipeanyiceorwaterfromsidesofcuvetteusingaKimwipe.Placethecuvetteintothesamplechamber.

                • Energizetheelectroporationapparatusanddeliverthepulsebypushinginbothchargingbuttonssimultaneouslyandholdinguntilashortbeepisheard.Notethetimeconstantofthepulseandtheactualvoltagedelivered.

                • Removethecuvettefromthesamplechamber.Immediatelyadd1mlSOCmediumandtransferthecellstoasterilepolypropyleneculturetubeusingaPasturepipette.
                  • FailuretoimmediatelyaddSOCtotheelectroporatedcellscansignificantlyreducecellviABIlityanddecreasetransformationefficiency.

                  • Incubateculturesfor30to60minutesat37°Conarollerorwithmoderateshakingtoallowforplasmidexpression.

                  • PlatealiquotsoftheelectroporationmixtureonL-agarplatessupplementedwiththeappropriateantibiotics.Incubateplatesat37°C.

                    SOCBroth(1liter)

                    Bacto tryptone 20.0 gBacto yeast extract 5.0 gNaCl 0.6 gKCl 0.5 gMgCl2 10 mM (see below)MgSO4 10 mM (see below)Glucose 20 mM (see below)

                    Note:SOCisidenticaltoSOB,exceptthatitcontains20mMglucose.

                    1. Dissolvetryptone,yeastextract,sodiumchloride,andpotassiumchlorideinafinalvolumeof970mldistilledH2O.Sterilizebyautoclaving.

                    2. Afterautoclaving,allowthesolutiontocoolto60°C,andadd20mlofasterile1Mglucosestock(seebelow)tomakethemedia20mMwithrespecttoglucose.

                    3. Justpriortousing,add10mlofmagnesiumstock(seebelow)totheSOCbrothtomakethemedia20mMwithrespecttomagnesium.

                       2MMg2+stock(100ml)1MGlucose(100ml)20.3gMgCl218.0gglucose24.7gMgSO4100.0mldH2O
                       DissolvereagentsinafinalvolumeDissolveglucosein90mldH2O.of100mldH2O.SterilizebyfiltrationBringtofinalvolumeof100ml.througha0.45µmdisposablefilter.SterilizebyfiltrationthroughaTheresultingsolutionis2Mwith0.22µmdisposablefilter.respecttoMg2+.