Purpose:

Toisolateintact,highmolecularweightDNAfromyeastcellsforsubcloningandrarecuttingrestrictionenzymeanalysis.Onecanexpectayieldof100-200礸ofDNAperprep.

Timerequired:

6daystotal

Day1-3:10minutesDay4:6-8hoursDay5:2hoursDay6:1hour

SpecialEquipment:

• 23mmdialysistubing

• BeckmanSW28RotorandUltracentrifuge

SpecialReagents:

• SCEsolution

• Lyticase(Sigma#L8137)

• Yeastlysisbuffer

• 15%,20%,and50%sucrosesolutions

Procedure:

Days1-3

1. Inoculateasingleyeastcolonyinto5-7mlofAHCmediumandincubateat30degreesCfor2days.Inoculate250-500mlofYPDmediumwiththeculture,andincubatewithshakingat30degreesCovernight.

Day4

1. Pourtheculturevolumeintolargecentrifugebottlesandcentrifugeat2500rpmfor15minutesintheBeckmanJ-6centrifuge.ResUSPendthecellpelletin40mlofdH2Obypipettingupanddownwhilescrapingthecellpellet.Transferto50mlconicaltube,andcentrifugeat2500rpmfor15minutes;decantsupernatent.Atthispointcellscanbefrozenforfutureuse,oronecanproceedasfollows.

2. Resuspendpelletin3.5mlofSCEwith30祃2-Mercaptoethanol(or40祃2MDTT)and10mgLyticase.Spheroplastat37degreesCfor1ormorehoursandshakegentlyevery15minutes.Continueuntilthesuspensionhasgreatlyincreasedinviscosity(oftenonecanseemanysmallbubbleswithintheviscoussuspensionatthispoint).

3. Pourthespheroplastsuspensionslowlydownthesideofa250mlflask(usedforthelargesurfaceareatheyprovide)containing7mlofLysisbufferwith100礸/mlProteinaseK.Takecaretogentlymixthesolutionandachieveuniformity.Placeflaskina65欳waterbathfor15minuteswithoccasionalshaking,thenrapidlycooltoroomtemperatureinawaterbath.

4. MakeacrudesucrosegrADIentbyfirstadding11mlof20%sucrose,then11mlof15%sucrose,thencarefullyunderlaying3mlof50%sucroseina36mlBeckmancentrifugetube(Ultra-Clear25x89mm).Slowlypourthelysateintothetube,thenultracentrifugeintheSW28rotorat26,000rpmat20欳forthreehours.

5. Aspiratethetopofthegradient(approximately30ml)untiltheviscousDNAonthebottomcanbeseentomove.Collectthisbottomlayerusinga10mlPipette,andplaceindialysistubing(23mm)thathasbeenpreviouslyprepared(seesectioninmanualonpreparationofdialysistubing).Attempttominimizethetotalvolume,forfluidwillbetakenupintothebag,dilutingtheDNAconcentration.Userubberbandsontheclipstopreventthemfromopeningunexpectedly.Placetubingina2literflaskcontaining1literofTE,addastirringbar,andplaceonastirplateat4degreesCovernight.

Days5-6

1. Removedialysisbagsandplaceonabedofdrysucrose,thencoverbagscompletelywithsucrose.Whenthesucrosebecomesdamp,replaceitwithdrysucrose;graduallyplacetheclipsnearertooneanotheronthedialysistubing,inordertoconcentratetheDNA.Thisprocessshouldtake30-90minutes.DialyzethebagsagaininTEovertheday,thenchangetheTEandallowtodialyzeagainovernight.

2. Takeofftheupperclipandplacethisportionofthedialysistubingwithina5mlsnap-captube.Draintheliquidintothetube.ItisveryimportantfromthispointontotrytominimizeshearingofthishighmolecularweightDNA;pipettingshouldbekepttoaminimumandshouldbedoneonlyusingtipswhichhavehadtheirendscutoff.Onecanexpectayieldof100-200礸ofDNApergradient.

3. ChecktheconcentrationoftheDNAbyrunningagainstknownstandards.DNAcanbereprecipitatedbyadding1/10volume3Msodiumacetateand2volumesofcold95%ethanol,thengentlyspoolingoutDNA;ifdonecarefully,littleshearingwilloccur.AddRNaseat50礸/mlwhenusingtheDNAinrestrictiondigests.

Solutions:

• SCE

  SCE		Finalconcentration
  2MSorbitol	50ml	1.0M
  1MSodiumcitrate	10ml	0.1M
  0.25MEDTA,pH7.0	24ml	60mM
  sterileddH2O	16ml
  	-------
  	100ml

  Filtersterilizeandstoreatroomtemperature.
  • LargeScalePrepLysisBuffer:
    0.5MTris-HCl,pH9.0

    3%Sarkosyl

    0.2MEDTA

    References:

    Carle,G.F.,andM.V.Olson.(1984)"SeparationofchromosomalDNAmoleculesfromyeastbyorthogonal-field-alterationgelelectrophoresis."NucleicAcidsRes.12:5647-5664.

    Burke,D.T.,Carle,G.F.,andM.V.Olson."CloningoflargesegmentsofexogenousDNAintoyeastbymeansofartificialchromosomevectors."(1987)Science236:806-812.