To enable screen reader support, press Ctrl+Alt+Z To learn about keyboard shortcuts, press Ctrl+slash
UzumakiSEA-PHAGEnotebookGroup#4AlexandruMedinaSameerBhattiFall2021TheSearchForSoil9/15Protocol5.1:CollectingEnvironmentalSamplesMaterialsneeded:●PlasticZiplocbagsforacquiringsoilsamples●SmartphoneortabletwithGPScapabilitiesorcomputerObjective:-Bothlabpartnershavetobringasampleofsoilfromarandomlocationoftheirchoosing-Informationlikethecoordinatesofbothsamples,descriptionsofthearea,andweatherconditionswererecorded.Alex’sSampleGPSLocation:40º43’21”N,73º53’33”WDescriptionofArea/Soil:Drysoilsurroundedbydebrisfromnearbyconstructionandlitter.WeatherConditions:80ºFSunnySameer’sSampleGPSLocation:40.70616°N,73.70895°WDescription:Moistsoilfromnearbywateredplants.WeatherConditions:82ºFSunnyDirectIsolation/FilteringProcess(½)9/15Protocol5.2:DirectIsolationObjective:ToextractphagesfromanenvironmentalsampleMaterialsneeded:●Environmentalsample●Liquidmedia*(5ml/sample)●Sterile3mlor5mlsyringe●0.22μmsyringefilter●5mlserologicalpipettes●Microcentrifugetubes●15mlconicaltubeVirusesaresmallerthanmicrobessowemixoursoilsamplewithliquid.Byusingcentrifugalforces(shakingit)wecanseparatethecomponentsthatarelargerthanviruses.ThisfilteringprocessisolatesthepotentialvirusesPlaqueAssay9/20Objective:DetectingthepresenceofphagesonbacteriallawnsSupplies:●Phagesamplesforisolation,purification,ortitering●Hostbacteria(250μl/plate)●Agarplates●Phagebuffer●Topagar,molten(between55-60˚C)●Microcentrifugetubes●5mlserologicalpipettesTheplaqueassayallowsyoutovisuallyconfirmthepresenceofphageparticlesinasample.Thepicturetotherightisanimageofoneofthemanybacterialawnsweused.Itisusedasabreedinggroundforourphages.ThebacteriawouldappeartobeafoggywhitesubstancealongtheplateProcedure1.Wepreparedourbenchandassembledallthesuppliesneeded2.Weinjectedthehostbacteriawithourphagesample.●Wetookthesameportionof250μlhostbacterialculturesaswehaveinthephagesamples,howeverwedidnotprepareitforapositivecontrolbutjustforonenegativecontrol.(Welabeledtheculturetubesaccordingly)●Thenusingamicropipette,wedispensedeachphagesampleintotheappropriateculturetubecontaining250μlofhostbacteria●Mixeachinjectedhostculturebygentlytappingthetube.Wethenletoursamplesitundisturbedfor10minutestoallowforattachment.Procedure4.MypartnerandIthenhadtotransfertopagarfromitsinitialjartothetubeswiththephageinjectedhostculture.Afterthatwehurriedlyglazedovertheinsideofourplatesbeforethetopagarhardenedandbecameunusable.a.Obtainthesamenumberofagarplatesasyouhavesamples.Sothismeantwelabeled6withoneofourinitials,thedate,andthespecifictubemixthatwentintotheplate.Inthispartoftheexperimentwedidnotcompleteanegativecontrol.b.Removeabottleoftopagarfromthe55°Cbath.c.Foreachsample,repeatinstructions:■Usingasterile5mlpipette,transfer3mloftopagartoaninoculatedhosttube(i.e.,thetubecontainingbacterialhostandphagesample).Wesuccessfullyavoidedmakingorwithdrawingbubbles,astheycanlooklikeplaquesonplates.■Immediatelysuck-upthemixturebackintothepipetteandtransferittotheappropriateplateanddiscardthepipette.■Gently,butquickly,tilttheplateinmultipledirectionsuntilthetopagarmixtureevenlycoatstheagarplate.5.Wethensetasidetheplatesandwaitedforourprofessortoorganizeourplates.Pickingaplaque9/20Protocol5.4:PickingaPlaqueObjective:ToretrievephageparticlesfromaplaqueandcreatealiquidsampleSupplies:●Agarplateswithplaquesofinterest●Phagebuffer●MicrocentrifugetubesAplaqueisazoneofclearingonabacteriallawnthatisformedwhenasinglephageparticleinfects,replicates,andlysedbacteria.Asaresult,aplaqueisfilledwithmillionsofidenticalphageparticles.Toretrievephagefromaplaque,aplaqueis“picked”andphageparticlesfromtheplaqueareresuspendedinphagebufferTheplaquewemadeconsistedaclearingonofmoltenagar,whichwouldbea“lawn”forthephagetoreston.Theclearingwouldbethephagesampletestingpositive.Procedure1.Firstlywepreparedourbenchforandassembledoursupplies.2.Labeltheplaques.1.Usingalabelingpen,marktheplaquesyouintendtopickbydrawingasmallcirclearoundtheplaqueonthebottomoftheplate.Ifpickingmultipleplaques,labeleachplaquewithauniqueletterornumber,orwithsomeotheridentifier.1.Itispossiblethatyouwillbepickingplaquesfrommorethanoneagarplate.Besuretolabelplaquesinawaythatwillallowyoutokeeptrackofthemandrecordthedetailsinyourlabnotebook.3.Recordthedetailedmorphologyofeachplaque(e.g.,size,cloudyorclear,margintype)youhavecircled.4.Labelandpreparemicrocentrifugetubes.1.Obtainasmanytubesasthenumberofplaquesyouintendtopick.2.Labeleachtubeaccordingtotheidentifieryouusedforeachplaque.3.Usingaseptictechnique,aliquot100μlofphagebufferintoeachmicrocentrifugetube.5.“Pick”aplaque.1.Placeasteriletipontoap200micropipette.2.Holdingthepipettorperpendiculartotheagarsurface,gentlystabthetopagarinthecenteroftheplaque(Figure5.4-1).1.Avoidtouchingthebacteriasurroundingtheplaque.3.Placetheendofthetipintothephagebufferinthecorrespondingmicrocentrifugetube.Thentapthetiponthewallofthetubeandpipetteupanddowntodislodgephageparticles.Discardthetip.4.Mixwellbyvortexing.5.RepeatSteps1–4foreachplaqueyouarepicking.Procedure(½)9/20Day11.YouwillneedthesolidenvironmentalsamplesyoucollectedusingtheprotocolCollectingEnvironmentalSamples(5.1).2.Extractphagesfromasoilsample.1.Filla50mlconicaltubewithyoursampletothe15mlmark.2.Addliquidmediatothe35mlmarkandvortex.3.Shakethesampleat~250rpmfor1–2hour1.4.Balancethetubesandcentrifugeat2,000xgfor10minutestopellet(i.e.,forcetothebottomofthetube)mostofthesoil2.3.Prepareyourbenchforasepticworkandassembleyoursupplies.1.Filterthesupernatantthrougha0.22µmfiltertoremoveunwantedbacteriaandsoilparticles3.1.CollecttheflowthroughinasterilebaffledErlenmeyerflaskora50mlsterileconicaltube.2.Recoveredvolumeswillrangebetween20and25ml.4.Seedtheculturewithhostbacteria.1.Add0.5mlofbacterialhostculturetotheflaskorconicaltube.2.Incubatetheflaskorconicaltubeatthepropertemperature,shakingat220rpmfor2–5days.1.Ifyouareusinga50mlconicaltube,youmustensurethattheculturewillbeproperlyaerated.Todoso,screwthecaponone-quarterofaturnsothattheconicaltubeisonlylooselycapped,andthensecurethecapwithashortpieceoflabtapetoensureitdoesnotfalloff.Checktomakesurethattheconicaltuberemainsonlylooselycapped.Tubesmustremainuprightwhilebeingshaken,andcaretakentoavoidspillage.2.Ifusingaliquidenvironmentalsample,youmustaddtheappropriatevolumeof10Xliquidmediaasasourceofnutrientsforyourhostbacteria.EnrichedIsolation9/20Objective:ToamplifyphagespresentinyourenvironmentalsamplesSupplies:●Solidenvironmentalsample●0.22µmCorning®Tube-TopVacuumFilterSystemsorsyringefilters●Liquidmedia*●10Xliquidmedia(ifusingliquidenvironmentalsamples)●BaffledErlenmeyerflask,Erlenmeyerflaskautoclavedwithpipettetipsinthebottom,or50mlsterileconicaltubes●Sterile5mlsyringes(ifneeded)●0.22μmsyringefilters(ifneeded)●Microcentrifugetubes●Hostbacteria(500μl)Weamplifiedourphagepresencebygivingitfavorableconditions.Weextracteditfromthesample,andmixeditintobacteriagrowthmedia.Thisfilteredsamplewouldallowthephagetoreplicateinourfavor.Procedure(2/2)Day2Aftertheenrichedculturehasbeenallowedtoincubatefor2–5days,youcancontinuewiththeprotocol.Prepareyourbenchforasepticworkandassembleyoursupplies.1.Filtertheenrichedculture.1.Usinganappropriatepipette,transfer1.4mlofyourenrichedculturefromtheErlenmeyerflasktoamicrocentrifugetube.2.Repeatthisproceduresothatyouhavetwomicrocentrifugetubes,eachwith1.4mlofenrichedculture.3.Spinthetubesathighspeedinthemicrocentrifugefor1minutetopelletthebacteria.4.Ifyoursupernatantisnotclearorifyoususpectyourenrichmentcontainsnon-hostbacteria,filterthesupernatantthrougha0.22µmfilterasdescribedbelow.Otherwise,proceeddirectlytoStep5.1.Removetheplungerfromasyringe.2.Openasterilefilterandattachittothebarrelofthesyringe.3.Pipette1mlofsupernatantfromeachmicrocentrifugetubeintothesyringebarrel(foratotalof2ml).4.Placethetipofthefilter/syringeoverasterilemicrocentrifugetubeandinserttheplungerintothesyringe.5.Depresstheplungerandcollectthesterilefiltrate.5.Transferthesupernatantintoacleanmicrocentrifugetube,avoidingthebacterialpellet.6.Immediatelycapthemicrofugetubecontainingyoursupernatantorfiltrateandlabelitappropriately.Itshouldbestoredat4°C.7.Eitherreturnyourculturetotheincubator,ordisposeofyourenrichedcultureasdirectedbyyourinstructor.2.Asdirectedbyyourinstructor,yournextstepwillbetotestyoursupernatantforphagesbyusingaSpotTest(5.6).SpotTest9/22Objective:TotestasampleforthepresenceofphagethatinfectedA.globiformis.Supplies:●Liquidphagesample(eitherapickedplaquefromadirectisolation,oranenrichedisolation)●Agarplates●Hostbacteria(250μl/plate)●Topagar,molten(between55-60˚C)●Phagebuffer●5mlserologicalpipetteForthisspottest,weadded6dropsofphage.Wedidn'tnecessarilyhaveto,sincetheyarefromthesamesample,butitprovidedabetterviewtoseeifourphagewaspositive.Allthespotsarethesamesample.Procedure(2/2)4.Spottheliquidphagesamplesandcontrolsonthepreparedbacteriallawn.1.Asepticallytransfer10μlofeachsample,oneatatime,ontotheproperlocationonthebacteriallawn.1.Holdthetipslightlyabovetheagarandexpelthedropslowlytoavoidsplattering.2.Besuretospotyoursamplesintherightplace!Rememberthatlabelsonthebottomofaplatearemirrorimages(i.e.,theywillappearbackward)ofyourlabelingschemewhentheplateisturnedover.2.Spot10μlofsterilephagebufferontheplateasanegativecontrol.3.Allowtheliquidfromthespotstoabsorbintotheagar(generally10–15minutes).4.Withoutinvertingtheplates,incubatethematthepropertemperaturefor24–48hours.5.Checkspotplatesforclearing.1.Afteratleast24hours,checkeachspotontheagarplate.Ifyouseeazoneofclearingforanyofyourspottedsamples,congratulations!Youroriginalsamplecontainedphage!2.MakesurethatyournegativecontroldoesNOTshowsignsofphages.6.Recordthedetailsofyourspotplateinyourlaboratorynotebook.7.YoucannowproceedtoChapter6,PurifyingYourPhage!PlaqueAssayforPurification9/27Protocol6.1:PlaqueAssayforPurificationObjective:Togeneratewell-isolatedplaquesSupplies:●Phagesamplesforpurification●Phagebuffer●Microcentrifugetubes●Hostbacteria●Agarplates●Topagar●5mlserologicalpipettesProcedure:Wepickedaphagethatwasfarenoughfromotherphagessowewouldn’tcrosscontaminate.Thenwedilutedourliquidphagesamples.After,welabeledour6platesandpreparedtotransfertopagarintotheplatemixedwithbacteriaandphage.Themorphologyofourphagesseemtobeclear.Therewerenootherkindsofmorphology.Thenumberofplaquesfollowtheexpectedpatternofourserialdilutions.FailedResult#1forfirstroundofdilutions9/29Ourfirstdayofworkingonthisprotocolendedinafailure,asourplatesbecamemessyduetoanerrorwhilefollowingtheprocedure.Themistakewasthatwedidn’tsecureenoughtopagarfortheexperimentandsoitdidn’tcovermostoftheplate.Thisresultedinanunreadableseriesofplates.Welearnedthatweneedtoensurethatwehaveenoughtopagarbeforetheexperimenttopreventanymoreerrors.Werestartedthisfirstroundofdilutionsthesamedayandtriedagainforthenextround.FailedPicture:Youcanseetheerrorinthelaterdilutions.SuccessfulResult#110/4Thenextdayresultedinapropersuccessfulresult,whichwaslegibleandshowedtheobjectiveclearly.SuccessfulPicture:Therearenoerrorsinthelaterdilutions,andthephagesarevisible.WeweresuperhappywecanmoveonSuccessful3rdroundpurification10/18Theresultsfromthe3rddilutionscameoutamazing,withclearvisibleclearings.WebbedplateThewebbedplateiswherewewillcollectthelysate,usedlaterinSpotTitering10/25FullPlatetiterToaccuratelycountthenumberofplaques,wearecompletinganotherserialdilutionspecificallyforsamples10^-3,10^-4,and10^-5becauseitfallsintherangeof20-200plaques.Afterthiswebeginanotherplaqueassayprocess.Wecounted48clearingsonthe10^-5plate,andthisfallsinthegivenrangeof20-200.Creatingspottitersforourduallysates11/8FirstwebegantwoseparateserialdilutionstocreatetwoseparatespottitersThismeansthatweshouldhavetesteddilutions10^7,8,andmaybe9.Sincewecouldn’tcalculatethetiterfromthespottiter,wemovedontofullplatetiters.3000lysatespottiterplate1500lysatespottiterplateFullplatetitersforbothlysates11/10Tomoveaheadtocalculatingthetiter,wehadtocreatefullplatetitersofbothlysates1500and3000sowecanaccuratelyseetheplaques.Wedidaserialdilutiontoafactorof10^-8,andonlyusedthe-6,-7,and-8dilutionfactors.Thiswillskipaheadtotheplateswithvisibleclearings.Wemixedthelysateswith300ulofbacteriainsteadof200,asthischangehadasuccessfulyieldearlier.Thesearethe3000lysateplatesshowingthedilutionsfor-6,-7,and-8.Thesearethe1500lysateplatesshowingthedilutionsfor-6,-7,and-8.11/15FullplatetiterresultsWeresuccessfullyabletocounthowmuchplaquesthereareontheplates.Sowecountedthe10^-6forboth1500and3000lysateplatetiters.The1500lysateplatetiterfor“-6”had90plaques,whilethe3000lysateplatetiteronlyhad74.Thereforeaftercalculatingthetitersforbothlysates,wefiguredoutthatthe1500lysateplatetiterhadthehighestyieldoftiter.Wefollowedthesameformulagiventoustocalculatethespottiterandacquiredthetitersforeachlysate.Thecircledworkaboveshowshehigheryield.DNAcleanup11/17TocleanupourDNAweinjected200ul100%ETOHand10ul3mNA-acetateintoourDNAsampleandinvertedtomix.Wethenputitiniceform30minutes.Thenwespunthemicrocentrifugetubeto10000rpmfor5minutes.WeremovedtheETOHbyinvertingthetube.WetriedtoremoveasmuchETOHaspossible.WethenfollowedthefollowingstepsAnimageofourPhagepelletonthesideofthetube.11/15OurDNAhadsomefaults.Thefollowinggraphshowsalinedeclinethenapeakthenadeclineagain.Thedeclineisnormalandthepeakat260showssignsofDNApresence.HoweverthelinestartingatthetopofthegraphshowssignsofDNAcontamination.SoinordertofixitwearegoingtohavetocleanuptheDNAbyprecipitatingitwithalcohol.ContinuationofDNAextraction11/15WashthesaltsfromtheDNA(nowinthecolumn)withthefollowingstepsforeachcolumn:Add2ml80%isopropanoltoeachsyringebarrel/columnandpushtheliquidthroughthecolumn.Repeattwice,foratotalofthreeisopropanolwashes.Removeresidualisopropanol.Witheachcolumninafresh1.5mlmicrocentrifugetube,spinat10,000×gfor5minutes.Thecolumnwillpreventthemicrofugetubelidsfromclosing.Arrangetheopentubesinthecentrifugesothatthelidspointtowardthecenteroftherotor.Transfercolumnstonew1.5mlmicrocentrifugetubes.Spinat10,000×gfor1additionalminutetoremoveanyresidualisopropanol.Evaporatethelasttracesofisopropanolbyremovingyourcolumnsfromthemicrocentrifugetubesandplacingthemdirectlyina90°Cheatingblockfor60seconds.ElutethephageDNAfromthecolumns.Placeeachcolumninacleanmicrocentrifugetubeandapply50μlof90°CsterileddH2Odirectlytoeachcolumn.Incubatecolumnsfor1minuteatroomtemperature.Spinat10,000×gfor1minuteinamicrocentrifuge.Combinetheproductsfrombothmicrocentrifugetubesintoonetube;thisisyourelutedphageDNA.Usingaspectrophotometer,quantifyyourphageDNA.11/15DNAextraction5mlof1500lysateinmicrocentrifugetubes.Wedidn’tusethe3000useitdidnothaveahighertiter.Thenourprofessoradded20ulofnucleasemix.Thenweincubatedthetubesin37°C.Wewaitedfor10min.Thenweplacedthe5tubesinahighspeedcentrifugeat10000xrpmfor1mintoseparatethesupernatantfromthephagepellets.WediscardedthesupernatantinatubeWemadesurenottosuckupanyofourphagepelletslocatedatthebottomofthemicrocentrifugetubes.Weusedthemicropipettewiththesettingon“500”ul.
HTML view of the presentation
Turn on screen reader support
Google Account
Alexandru Medina
amedin09@nyit.edu