Cellsareroutinelypassagedtwodayspriortoelectroporating.Usuallyone10cmplateatapproximately80%confluencywillprovideenoughcellsfor1-2electroporations. Procedure 1.ChangemediumonEScells3-4hourspriortoelectroporation 2.Gelatinize10cmplates,thenadd10mlmediumtoeach. 3.Placethemina370Cincubatoruntiltheyarerequired. 4.Switchontheelectroporationapparatus. 5.HarvestEScellsbytrypsinization. 6.ResUSPendthecellpelletinice-coldPBS(1mlforeach10cmplate). 7.Determinethecelldensity(haemocytometer)anddilutewithPBStotherequireddensityforelectroporation.Weregularlyelectroporateatarelativelyhighcelldensity:7x106cells/ml(thisnumbervariesbetweendifferentlabs). 8.Foreachelectroporationmixtogether20-40microgram(1礸/祃)DNA(foranapproximately10kbvector)and0.8mloftheEScellsuspensioninanelectroporationcuvette(BioRad,Cat.No.165-2088). 9.Setuptheelectroporationconditionspriortoplacingthecuvetteintotheelectroporationchamber.Weroutinelyuse250V,500microFfortheBioRadGenePulser. 10.Zapthecuvette,thenplaceitonicefor20minto1hour. 11.Transferthecellsfromthecuvetteintotheprewarmedmediumcontainingdishes.(Thecontentsofonecuvetteareroutinelyseededintotwo10cmdishes). postelectroporation: 12.Changemediumdaily. 13.Ifdrugselectionisrequiredstartthisontheseconddayafterelectroporation. 14.Continuetheselectionuntilcoloniesbecomeapparent,andgrowtoasizethatisamenabletopicking(usuallytakes7-10days).