1.UseafreshcolonyofDH5α(orotherappropriatehoststrain)toinoculate5mlofSOB(withoutmagnesium)mediumina50mlsterileconicaltube.Growcellswithvigorousaerationovernightat37EC.

2.Dilute2.5mlofcellsinto250mlofSOB(withoutmagnesium)ina1literflask.Growfor2to3hourswithvigorousaerationat37ECuntilthecellsreachanOD₅₅₀=0.8.

3.Harvestcellsbycentrifugationat5000RPM(2,600g)inaGSArotorfor10min.(Makesureyouuseautoclavedbottles!).

4.WashthecellpelletbyresUSPendingin250mlofsterileice-coldWB(10%redistilledglycerol,90%distilledwater,v/v).Centrifugethecellsuspensionat5,000RPMfor15minandcarefullypouroffthesupernateassoonastherotorstops.CellswashedinWBdonotpelletwell.Ifthesupernateisturbid,increasethecentrifugationtime.

5.Washthecellpelletasecondtimebyresuspendingin250mlofsterileice-coldWB.Centrifugethecellsuspensionat5000RPMfor15minandpouroffthesupernate.

6.ResuspendthecellpelletinWBtoafinalvolumeof1ml.UsuallynoadditionalWBneedstobeaddedtothecellpellet;itcanberesuspendedintheWBthatremainsinthecentrifugebottle.Cellscanbeusedimmediatelyorcanbefrozenin0.2mlaliquotsinmicrocentrifugetubesusingadryice-ethanolbath.Storefrozencellsat-70°C.

Forbestresults,DNAforelectrotransformationmusthaveaverylowionicstrengthandahighresistance.TheDNAmaybepurifiedbyeitherprecipitationordialysis.

PurifyingDNAbyPrecipitation:

1.Add5to10μgoftRNAtoa20μlligationreaction.Add22μl5Mammoniumacetate.Mixwell.

2.Add100μlabsoluteethanol.Ice15min.

3.Centrifugeat>12,000xgfor15minat4°C.Carefullydecantthesupernatant.

4.Washthepelletwith1mlof70%ethanol.Centrifugeat>12,000xgfor15minatroomtemperature.Removethesupernate.

5.Airdrythepellet.

6.ResuspendtheDNAin0.5XTEbuffer[5mMTris-HCl,

0.5mMEDTA(pH7.5)]toaconcentrationof10ng/ulofDNA.Use1μlpertransformationof20μlofcellsuspension.

1.Marktherequirednumberofmicrocentrifugetubes.PlacetherequirednumberofMicro-electroporationChambersonice.FillthetemperaturecontrolcompartmentoftheChamberSafewith~250mlofice-waterslurryandplacetheChamberRackintheChamberSafe.

2.ThawanaliquotofcellsthathavepreparedasinSectionIandaliquot20Flofcellstotherequirednumberofmicrofugetubesonice.Add1FloftheDNA(orligationreaction)preparedasinSectionII.

3.UsingamicroPipette,pipette20Flofthecell-DNAmixturebetweenthebossesinaMicro-ElectroporationChamber.Donotleaveanairbubbleinthedropletofcells;thepressureofabubblemaycausearcingandlossofthesample.PlacethechamberinaslotintheChamberRackandnoteitsposition.Repeattheprocessifmorethanonesampleistobepulsed.Upto4samplescanbeplacedintheChamberRackatonetime.Handlethechambersgentlytoavoidaccidentallydisplacingthesamplefrombetweenthebosses.

4.ClosethelidoftheChambersafeandsecureitwiththedrawlatch.

5.PlugthepulsecableintotherightsideoftheChambersafe.

6.TurnthechamberselectionknobontopoftheChamberSafetodirecttheelectricalpulsetothedesiredMicro-ElectroporationChamber.

7.SettheresistanceontheVoltageBoosterto4kΩ;setthePulseControlunittoLOWand330FF;doublecheckconnections.

8.ChargethePulseControlunitbysettingtheCHARGEARMswitchonthePulseControlunittoCHARGEandthenpressingtheUPvoltagecontrolbuttonuntilthevoltagereADIngis5to10voltshigherthanthedesireddischargevoltage.ForE.coli,thestandardconditionsare2.4kv,whichmeanssettingthePulseControlunitto405volts(400voltsisthedesireddischargevoltage+5).Thevoltageboosteramplifiesthevoltsby~6-foldsuchthatthetotaldischargevoltageis2400volts,or2.4kv.TheactualpeakvoltagedeliveredtothesamplewillbeshownontheVoltageBoostermeterafterthepulseisdelivered.

9.SettheCHARGE/ARMswitchtotheARMposition.ThegreenlightindicatesthattheunitisreadytodeliveraDCpulse.DepressthepulsedischargeTRIGGERbuttonandholdfor1second.

NOTE:TheDCvoltagedisplayonthePulseControlunitshouldread<10voltsafterapulsehasbeendelivered.Ifnot,dischargethecapacitorusingtheDOWNbutton.

10.Foradditionalsamples,turnthechamberselectionknobtothenextdesiredpositionandrepeatsteps8and9untilallsamplesarepulsed.

11.Forampicillinselection,inoculatethesamplesinto2mlofSOCmediumandshakefor60minutestoallowexpressionoftheβ-lactamasegene.PlatecellsonLBmedium+50μg/mlampicillin(andX-galandIPTG,ifappropriate).