TransformationofGram-PositiveBacteriaanadaptationfromChang,D.,Chassy,B.,Saunders,J.,Sowers,A.1992.GuidetoElectroporationandElectrofusion,AcademicPress,Inc.,SanDiego,CA.Guidingprinciples:keepthecellsascoldaspossIBLeatalltimes;manipulatethecellsasgentlyaspossibleatalltimes.1、MakingelectrocompetentgrampositivebacteriaStartafreshovernightculturewiththebacteriaofinterest.Cellsshouldbeharvestedatearlytomid-exponentialgrowthphase.Harvestthecellsbycentrifugation.Moststrainsshouldremainchilledduringtheentireprocedurefromthepointofharvest.Chooseagrowthmediumforsatisfactorygrowthundernormalconditions.Themediummaybeoptimizedtoyieldthebestefficienciespossible.Mediadefinedasrich,limiting,defined,orminimalshouldbeconsidered.Othervariablesaffectingthegrowthofthebacteriamayalsoconsideredsuchastemperature,substrates,andadditions.Itisimportanttowashthecellsthoroughlytoremoveanymediumorelectrolytesthatmaybepresent.Washingcellsthreetofourtimesisadequate.Themediumchosentowashthecellsanduseastheelectroporationbuffershould1)keepthecellsviable,2)havehighelectricalresistance.Highqualitydeionizedwaterworkswellinmanyapplications.Additivessuchas0.27-1.0Msucrose,10-15%glycerol,1Msorbitol,PEGandsimilarreagentsmaybeusedtoactas"osmoticstABIlizers"orcryprotectants.Insomeinstances,theseadditiveshelptoincreaseefficiencies,inothers,theyinterfer.BufferscontainingHEPES,MOPS,orphosphatearelowresistancebuffersandmustbeavoided.Magnesiumandcalciumchlorideshouldalsobeavoided.ResUSPendthewashedcellsinelectroporationmediumtoafinalconcentrationof5x10exp9to5x10exp10cells/ml.Keeponiceuntiluse.Manystrainswillremainelectrocompetentforhoursandcanbefrozenforfutureuse.Quickfreezethecellsinandryicebathandstoreat-70°C.2、ElectroporationofCellsThepulseparametersshouldbemadebyconsultingspecificreferencesforthegenusandstrainbeingtransformed.Ifspecificliteraturecannotbefound,evaluatetheparametersforcloselyrelatedspecies.Thecontrollingparametersarefieldstrength(kV/cm)andtimeconstant(tau,ms).Typically,cocciwillrequirehigherfieldstrengths(7-18kV/cm)andthebacillirequirelowerfieldstrengths.Preliminaryexperimentsshouldbeperformedtodeterminetheoptimalfieldstrength.Optimaltimeconstantsforgram-positivebacteriaarereportedtobebetween2.5and7.5ms.MixtheDNAofinterest(0.1-1μgin1-2μlofwater)andthebacteria.Placethesampleintoaprechilledcuvette.Becertainthesampleisincontactwithbothsidesofthecuvettebytappingthecuvettelightlyonasolidsurface.Choosethecuvettesize(1or2mm)inaccordancewiththefieldstrengthamdsamplevolumedesired.Samplesizewillbedeterminedbythecuvettechosen.Insertthecuvetteintotheinstrumentanddeliverthepulse.Addmediadirectlytothecuvetteandplatealiquotsonselectivemedia.Incubateundernormalconditions.Itmayenhancetransformationtoallowthecellstorecoverinanon-selectivemediaforaperiodoftime(typicallyanhour)beforeplating.Thisallowsthecellstorecoverfromtheelectricpulseandtoallowthemtoexpresstheantibioticresistance.Afterrecovery,platealiquotsonselectivemediaandincubateundernormalconditions