GeneKnockoutwithConventionalMutagens

LeonAvery

ENG (enriched nematode growth medium): 1. in a 6 L flask add:3800 ml DH2O20 g. bactopeptone4 g yeast extract12 g. NaCl4 ml 5mg/ml cholesterol in EtOHstir with a large stir bar until dissolved.2. Add 120 g. agar and stir well.3. Cover w/ Al foil, autoclave liq. cycle 30" (turn on the 70C water bath before the agar is autoclaved)4. Equilibrate temp. in water bath.5. Just before pouring the plates add (sterile solutions) with stirring:4 ml 1M CaCl24 ml 1M MgSO4100 ml 1M KHPO4 (pH6) 4 ml 200 mg/ml streptomycin1 ml 40 mg/ml nystatin in DMF (add dropwise)AENG (agarose ENG):Same recipe as ENG, except replace 3% agar with 2% agarose, and youprobably won"t want to make so much.Making a grid:1. Inoculate one or more 10 cm ENG plates with 10 N2 L4 hermaphrodites. Four or five days later, when there are many gravid adults, prepare eggs by the alkaline hypochlorite method. Leave the eggs in M9 after the final wash.2. Put the tube of eggs in M9 on a rocker (or something else that will aerate them) overnight.3. Count the healthy L1s in a 10 ul aliquot of the sUSPension.(Iusea10ulmicrocaptospreadthemonanunseeded6cmplate.)Basedonthecount,put5000healthyL1soneachof1010cmENGplates.ThesewillbeyourP0s.4.WhentheP0shavereachedL4,harvestthemin3mlM9.(You"llprobablyhavetospinthemdownandtransferthemtonewM9togetthevolumethatlow.)5.Dissolve20ulEMSin1mlM9.AddthissolutiontoyourP0s.6.Aeratefor4hours.7.Spindown,transferto4mlfreshM9.8.Put5000P0soneachof1010cmENGplates.Allowthemtobecomegravidadultswithlotsofeggs.(Idothisat15C,sincetheL4swillbecomegravidadultsinconvenientlysoonat20C.)9.Harvestalltheplatesandprepareeggsbyalkalinehypochlorite.Aerateovernight.10.CountthehealthyF1L1sina10ulaliquot.Therewillbealotofsickordeadones(theirparentsweremutagenized)--don"tcountthese.DeterminingnumbersofF1s11.Put1250F1L1soneachof1106cmAENGplates.F1sperplate12.After3daysat20CmostoftheF1sshouldbegravidadults.Countthegravidadultsononeortwoofyourplates.Thereshouldbeabout1000(lessthan1250,becausemanyoftheF1sdidn"tgrowuporweresterile).F1sperplate13.Afteranother2days(5daystotal)theplateshavestarved,andalmostallthewormsonthemareL1s.Harvest96oftheplateswith5mlM9each.Discardanyextraplates--they"rejustincasesomeofyourplatesgotmoldy,orotherwisedidn"twork.Harvestingtheplates14.Counta10ulaliquotfromafewofthetubes.Youshouldhaverecoveredbetween100,000and200,000F2L1sfromeachplate.F2sperplate15.Letthetubessitonyourbenchuntilthewormshavesettledtothebottom.withaPasteurpipetattachedtovacuum,suckoffliquidsothatyouhaveabout1.5mlleftineachtube.(ThisisthestepIworryabout--I"mafraidifthewormssitatthebottomofthetubetoolongtheymaygoanaerobicanddie.Idoplatesinbatches,sothattheydon"thavetosittoolong,andmixthetubesfromtheearlierbatchesoccasionallywhileworkingonthelaterones.)16.Forthisstep,Iusesterile1.2mltubesinan8x12microtiter-spacedrack.Youwantthreeracksof96,twoforwormstoberecoveredlater,andoneforDNApreps.Put0.5mlof2xfreezingsolutionineachofthetubesofthetwowormracks.Then,forthefirstofyour96tubes,put0.5mlintotheA1positionofeachofthethreeracks.Forthesecond,put0.5mlintotheA2position,etc.17.Putthetwowormracksintoastyrofoamboxina-80Cfreezerandleaveovernight.YoucanfreezethewormsfortheDNAprepsnow,orgoaheadanddotheDNAprepsimmediately.18.Letthewormssettletothebottomsofthetubes,andsuckoffalltheliquidyoucan.DNApreps19.Add250ulfreshlysissolution(200mMNaCl,100mMTris-HClpH8.5,50mMEDTA,0.5%SDS,100ug/mlproteinaseK)toeachtube.20.50C,1h.(ThisisshorterandcoolerthanthetypicalincubationsusedforDNApreps.Itworksfine,andlongincubationsathighTaremutagenic.)21.PrepareDNAfromeachtubebyphenol:sevagextraction,sevagextraction,andEtOHprecipitation.Redissolvein150ulTER(10mMTris-HClpH8,1mMEDTA,1ug/mlRNAaseA.Storeat-80C.22.Use2ulof1:5dilutedDNAineachdetectionreaction.Mutationdetectionprotocols:Ihaveusedthreedifferentprotocolsfordetectingmutations.Thefirstisfordetectingpointmutationsinarestrictionsite.Iwon"tdescribethis,sinceIdon"tcurrentlythinkit"sthebestwaytoknockoutagene.ThesecondmethodisthatcommonlyusedfordetectingdeletionofaTc1byrelyingonthemuchgreaterefficiencyofstandardPCRinamplifyingshorterfragments.Icallthismethod"primerapproximation",sinceitreliesonthedeletionapproximating(bringingclosetogether)theprimers.Thethirdmethodasksforthedeletionofaclusterofrestrictionenzymesites.Ithasthetheoreticaladvantagethatitmaydetectsmalldeletionsthatdon"tmuchchangetheefficiencyofamplification.Whetherthisisapracticaladvantageisnotclear.Idonotusenestedprimers.Ithinkthisreducesthefalsepositiverate,butitdoesmeantheprimershavetobeprettygood,sinceyouneedtobeabletoamplifyfromaslittleas10moleculestoagoodstrongband.Therefore,beforescreeningwithanyprimerpair,Ifirstrunaseriesofreactionsinoculatedwith0,10,1000,and100,000wild-typegenomes,andrununderlongPCRconditions(basicallytheconditionsforsitedeletionbelow).Imake25-merprimerswith40-60%GC,usingtheWhiteheadInstituteprimerprogramtohelpfindthemandavoidprimer-dimers,etc.Mostofthepairswork.Mutationdetectionbyprimerapproximation:SolutionA:0.4ul5U/ulTaqpolymerase5ul10xPCRbuffer(100mMTrispH8.3,500mMKCl,20mMMgCl2)0.5ul10mg/mlacetylatedBSA(NEB)1ul10uMleftprimer1ul10uMrightprimer1ul10mMdNTPs(10mMeachofdATP,dCTP,dGTP,dTTP)39.1ulwater-------48ulPut48ulAineachof96thin-wall200ultubes.Add2ul1:5dilutedDNAtoeachtube.RunPCRprogram(MJRPTC-200):Controlmethod:CALCULATED1:92degreesforever,beep.2:92degrees,55sec.3:92degrees,5sec.4:65degrees,30sec(adjustfortheTmofyourprimers).5:68degrees,1min.6:gotostep3for39morecycles.7:68degrees,4min.8:4degreesforever,beep.StartthePCRmachineandwaitfortheblocktoheatup.Nowputthetubes(keptonice)intheblock.Thesolutioninthin-walltubeswillheatupveryfastwhenplacedinapreheatedblock:thisisalmostasgoodasahotstart.PushtheproceedbuttononthePCRmachineonceallthetubesareloaded.Add10ul6XgelloADIngbufferandrun12ulon1%gel.Mutationdetectionbysitedeletion:Inthisexample,theenzymeBstBIisused.YouneedtouseaThermophilicenzyme,sincecoolingthereactiondownto37CfortheseconddigestionhasdisastrouseffectsonPCR.YoualsowantanenzymethatwillworkprettywellinPCRbuffer,andthatisprettycheap.ThereareawholeseriesofenzymesfromBacillusstereothermophilusthatmeetthesecriteria.SolutionA:0.25ul20U/ulBstBI,NEB1ul10xNEBuffer40.1ul10mg/mlacetylatedBSA,NEB6.65ulwater------8.0ulSolutionB:0.01ul5U/ulPwopolymerase0.09ul5U/ulTaqpolymerase3.5ul10xPCR-NEB4(75mMtrispH8.6,500mMKCl)0.35ul10mg/mlAcBSA1ul10uMprimer11ul10uMprimer21ul10mMdNTPs28.05ulwater--------35.0ulSolutionC:0.3ul5U/ulTaqpolymerase0.5ul20U/ulBstBI0.5ul10xPCR-NEB40.05ul10mg/mlAcBSA3.65ulwater-------5.0ulLAMD45PCRprogram(CALCmode,MJRPTC-200):192Cforever,beep292C,1min365C,30sec(annealingtemp)468C,5min565Cforever,beep(Tinsteps5&6isdigestionTforenzyme)665C,30min792C,5sec865C,30sec968C,2min10gotostep7for8morecycles1192C,5sec1265C,30sec(annealingtemp)1368C,2min+10sec/cycle14gotostep11for34morecycles1572C,10min164Cforever,beep1.Mix2ulDNAsolution(intendedtobeabout100,000genomes)with8ulA.Incubate65C,10min.2.Add35ulBtoeachtube.Keepeverythingonicewhileyoudothis.3.StartthePCRmachine.Whenitreaches92C(step1),transferthetubesfromtheicetotheblock.Onceallthetubesareintheblock,tellthemachinetoproceed.4.Atstep5,themachinewillhaltagainat65C.Leavingthetubesintheblock,add5ulCtoeach.Whenfinished,tellthemachinetoproceed.6.Runon1%agarosegel.