ProtocolforextractingDNAfromESCells,startingfromthe96-wellplatebutprocessinginanEppendorftubetorecovermoreoftheDNA.NOTE-THISTAKESALOTOFTIMEifyoudothewholeplatethisway!

Lysisbuffer:(100mlRecipe)10mMTris-HClpH7.5-0.5mlof2M10mMEDTA-2mlof0.5M10mMNaCl-0.2mlof5M0.5%(w/v)Sarkosyl-0.5gm(N-Lauroylsarcosine,Sigma#L-9150)1mg/mlProteinaseK-addfresheachtime(storedinfreezer).

1.Add250µlLysisBuffer(withproteinaseKadded)toeachwell.

2.Incubatetheplateinasealedhumidcontainerat60°ree;Cfor1hour.(Meanwhile,labeleppytubes!)

3.Transferthecontentsofeachwellintoaseparate1.5mleppendorftube(µ-cent.tube).

4.Continuelysingthecellsfor2morehours.(60°ree;C).(Floattubesinwaterbath-don"tneedtupperware).

5.Addanequalvolume(250µl)ofphenol:chloroform(phenol:chloroform:isoamylalcohol,25:24:1).(ThepHofthephenol:chloroformneedstobe~7.8-8.0,orelsetheDNAwillpartitionintotheorganicphase).

6.Mixthecontentsofthetubeuntilanemulsionforms.(Shakebyhandfor~1min,(orvortex~2sec),becausevortexingshearslong(genomic)DNA).

7.Centrifuge5-10mininamicrofugeatTRm.(Thephasesshouldbewell-separated).

8.Transfer(pipet)theaqueousphase(thetopphase)toafreshtube.(Tosstheinterface/organicphase--chemicalwasteinhood).

9.Repeatsteps5-8untilnoproteinisvisIBLeattheinterfaceoftheaqueousandorganicphases.

10.Addanequalvolume(250µl)ofchloroformandrepeatsteps6-8.

11.Optional:Repeatchloroformextraction.(Thechloroformextractionremovesthetracesofphenol).

12.Add1/25thvolumeof5MNaCl(10µlof5MNaClfor250µl),forafinalconcentrationof0.2MNaCl.Mixwell.(useNaClratherthanothersaltsbecauseofdetergentinlysisbuffer).

13.Addexactly2volumesofice-coldethanolandagainmixthesolutionwell.

14.Storetheethanolicsolutiononicefor30mintoprecipitatetheDNA.(canbestoredindefinitelyat0°ree;Corat-20°ree;C).

15.Centrifugeat12,000xgfor10min,0°ree;C.(Try4°ree;C,microfugeatmax(16,000),10min).(Remembertopointtubehingesout,tolocatepelletevenifit"sinvisible).

16.Suckoffthesupernatant(dispopipettip,withvacuumflask).Donotdisturbthepellet.Vacuumdropletsfromwallsoftubeaswell.

17.Halffillthetubewith70%EtOHandre-spinfor2minutesat4°ree;C.

18.Suckoffsupernatantasinstep16.

19.StoretheopentubeonthebenchatTRmuntilthelasttracesoffluidhaveevaporated.(i.e.:Airdry).

20.DissolvetheDNAin30µl(or50µl,andusehalfofsample)of10mMTrispH8.5(orTE).(pipetaroundthewallsofthetubetodissolvetheDNAoffthewallsofthetube).Letdissolveforatleastanhourat37°ree;C.

21.Restrictiondigest:-DNAinTrispH8.5,fromaboveprotocol-restrictionenzyme-restrictionenzymebuffer-X(BSAifrequiredbyenzyme)-spermidine(toafinalconc.of1mM;storedinfreezer)-(alsoadd:)DTTtoafinalconc.of1mMFinalvolume=40µlorless.Digestovernightat37°ree;C.

Next:Rungels;Southernblotandhybridize.ShirleyReynolds