Need1.5-2x107cellsfroma2dayculture.

1.Cellsareharvestedasnormal,washedx1inPBSthentakenupatconc.of1.2x107cells/mlincoldPBS.

2.Takex2Bioradcurvettes

A.0.9ml(107cells)

B.0.167mlcells(2x106cells)+0.73mlPBS

ToAadd15uglinearisedplasmidDNA,IMMEDIATELYpriortoelectroporation..

BnoDNA

3.UsingaBioradGenePulser,(McMahon,A.P.,andBradley,A.(1990)Cell621073-1085).

"Zap"eachcurvettetwicei.e.500uF230Vthen500uF240V

4.ASAPusingapasteurPipettetransfercellsfromcurvette

Atoa10mltubecontaining4mlsmedium,thenplate5x1mlcellsontofive10cmdiapetridishescontainingNeoPMEF"sand10mlmedium.Labelplates4-8.

Btoa10mltubecontaining1mlmedium,thenplateallontoa10cmdiapetridishcontainingNeoPMEFsand10mlmedium.Labelplate2.

5.Setupfollowingcontrolplates.

a)Take0.167ml(2x106cells)originalcellsUSPension_10cmdiapetridishcontainingNeoPMEF"sand10mlmedium.Labelplate3.

b)Take0.167ml(2x106cells)into10mlmedium(~2x105cells/ml).Plate0.1ml(2x104cells)ontoNeoPMEF"sin10cmpetridishwith10mlsmedium.Labelplate1.

6.AllplatesincubatedinCO2incubatorovernight.

7.Nextdayapprox.24hrsafterelectroporationplatesreceiveappropriateselectionmedium.

*NBifplasmidcontainsaTKpromoterandpositive,negativeselection(PNS)isusedtheseplatesalsoreceiveGancyclovirorFIAUaswellasG418.

Mediumischangedonallplatesevery48hrs.By3daysnonG418resistantclonesshouldbedyingoff,afteroneweekthereshouldbenoclonesonPlates2&3.Ifthereareclonespresenttheexperimentisinvalid.After6-7daysthecoloniesonplates5-8(plates6-8ifPNSisused)arereadyforharvesting.Forefficientpickingofsingleclonesthereshouldbenomorethanapprox.100colonies/plate.

Pickingofsinglecolonies

PlatesaretreatedoneatatimeandasquicklyaspossIBLe.RemovemediumwashwithPBS.Don"twashwithPBS/EGTAasthiscausesthefeederstoliftoffandtheEScellcoloniestofloatawayandbreakup.

AddasmallvolumeofPBStoplate,holdplateoverapieceofblackcardboard,usingaP-200GilsonpickcolonybringingwithitthesmallestpossiblevolumeofPBS.Pick6coloniesinsuccessionintoa96wellplatecontaining20ulTRYPSIN/EDTA.Checkusingmicroscopethattherearesinglecoloniesineachwell.Usingamultichannelpipettecoloniesaredispersedbypipettingupanddown40xinacircularmotionbeingcarefulnottomakebubblesasthesecausecelldeath,checkthatcoloniesaredispersed.Cellsarethentransferredtoa96welltray,containingNeoPMEF"s.Thisprocessisrepeateduntilallcloniesarepicked.After3-4daysclonesshouldbereadyforsplittingandfreezingdown.

SplittingandFreezingdownofclones

Treatingonerowatatime(theremaybeclonesthatarenotready),removemediumusingaspiratorandsterilepasteurpipette,washeachwellwith0.1mlPBS,aspirate,washwith0.1mlPBS/EGTA,aspirate,add40ulTRYPSIN/EDTA,usingamultichannelpipettedispersecellsasbefore,transfer25ulinto96wellplatecontaining10%DMSOinmedium.ThenusingaGilsonandasteriletiptransferremainingcellstoa24wellplatewith1mlmediumwithLIF(nofeeders)ineachwell,24wellsarepre-treatedwith0.5mlgelatin/PBS.TheseculturesareforDNAisolationforSouthernanalysis.

Onceentiretrayiscomplete,the96welltrayistapeduparoundtheedge,occupiedwellsarerecordedonrecordsheetandtrayisthenputintoapre-chilledfoamboxat-70.Onceculturesarefrozen>2hrsthetraysaretransferredtoaneskyat-80andstoreduntilSouthernanalysisiscomplete.

IsolationofDNAfrom24Wellplates

Onceculturesarealmostcofluent,(itdoesn"tmatterifthecoloniesareverylargeorifsomeofthecellshavedifferentiated,asthiswon"teffecttheDNA),theyarereadyforharvesting.

1.Removemediumusingaspiratorandpasteurpipette.

2.Washeachwellx2with0.5mlPBS.

3.Add0.2ml6MGuanidineHCl/NaAcsolution,cellslysealmostimmediately.

4.Usingapasteurpipetteandarubberteat,slowlysuckuptheviscoussolution.ItisimportanttodothisslowlyotherwisetheDNAwillshear.TransferthesolutionintoalabelledEppendorftube.Thetubesarethentapedintoaneppendorfrackandtapedtothewheeltomixovernight.

5.Nextday,inlotsof12or20dependingonsizeofrackused,add500ulAbsEtOHtoeachtubeusingapipetteaidanda5mlpipette.Mixeachtubebyinversion(don"tvortex)untilDNAprecipitates.

6.Prepareaseriesoftubescontaining250ul70%EtOH,thiscanbedispensedusingmultipetteeppendorf,andalabelledserieswith200ulTEineach,alsodispensedusingthemultipetteeppendorf.

UsingaglasscapillarytubeandholdingyourforefingerovertheendtoavoidtransferringEtOHalongwiththeDNA,scoopouttheDNA,stillholdingfingerovertheendofthetubedipDNAinto70%EtOH,wrigglearoundtowashtheDNAthentransferDNAintotubecontainingTE.DNAshouldcomeofftheendjustbywrigglingthetubing,ifnotclosethelidontotheglassandsnapofftheexcess,leavingtheDNAattachedtotheglass.

NB.ThetransferoftheDNAbetweentubesmustbedonequickly,especiallythestepbetweentheAbsEtOHandthe70%EtOHastheDNAdriesoutveryquicklyandisthenvirtuallyimpossibletoredissolve.

7.TodissolvetheDNA,tubesareplacedinrackstapedtothebottomoftheshakerincubatorinthe32Plabandshakenovernightonspeed10@50.Thesolutionshouldbeslightlyviscous,theremaybesomeundissovleddebrisinthetube,thiswillbreakupbypipettingitupanddown.Itdoesnotinterferewiththerestrictionenzymedigestion.

8.75ulofdigestedDNAisenoughforSouthernanalysis.ThisleavesenoughforarepeatSouthernifnecessary.

G418(Geneticin,Gibco-BRL11811-031)need150ug/mlactive(however,eachbatchshouldbetested.MakeupX100inPBSandfiltersterilize.

NB.Eachbatchhasnumberofugs/mgactivewrittenonvial,youneedtoallowforthiswhenmakingyourcalculation.

FIAU(1-[2-deoxy,2fluoro-?D-arABInofuranosyl]-5iodouracil)

Ineppendorfsin-80(Madeupas2000X)

Workingconcentration=0.2uM.

Gancyclovir.FreezedriedpowderformSyntex.$57.71/vialRMHpharmacy.

6MGuanidine-HCl,0.1MNaAc(pH5.5)

57.36g/100mls

add1/303MNaAcpH5.5