58.456(mwofNaCl)gx5molesx0.1liter=29.29gin100mlsolmoleliter

Ex.Tomakea0.7%solutionofagaroseinTBEbuffer,weight0.7ofagaroseandbringupvolumeto100mlwithTBEbuffer.

Manyenzymebuffersarepreparedasconcentratedsolutions,e.g.5Xor10X(fiveortentimestheconcentrationoftheworkingsolution)andarethendilutedsuchthatthefinalconcentrationofthebufferinthereactionis1X.

Ex.Tosetuparestrictiondigestionin25μl,onewouldadd2.5μlofa10Xbuffer,theotherreactioncomponents,andwatertoafinalvolumeof25μl.

Ex.Tomake100mlofTEbuffer(10mMTris,1mMEDTA),combine1mlofa1MTrissolutionand0.2mlof0.5MEDTAand98.8mlsterilewater.Thefollowingisusefulforcalculatingamountsofstocksolutionneeded:

C_ixV_i=C_fxV_f,whereC_i=initialconcentration,orconcofstocksolution;

V_i=initialvol,oramountofstocksolutionneeded

C_f=finalconcentration,orconcofdesiredsolution;

V_f=finalvol,orvolumeofdesiredsolution

1. Refertothelaboratorymanualforanyspecificinstructionsonpreparationoftheparticularsolutionandthebottlelabelforanyspecificprecautionsinhandlingthechemical.
2. Weighoutthedesiredamountofchemical(s).Useananalyticalbalanceiftheamountislessthan0.1g.
3. Placechemical(s)intoappropriatesizebeakerwithastirbar.
4. Addlessthantherequiredamountofwater.Prepareallsolutionswithdoubledistilledwater(incarboy)
5. Whenthechemicalisdissolved,transfertoagraduatedcylinderandaddtherequiredamountofdistilledwatertoachievethefinalvolume.Anexceptionisinpreparingsolutionscontainingagaroragarose.Weightheagaroragarosedirectlyintothefinalvessel.
6. IfthesolutionneedstobeataspecificpH,checkthepHmeterwithfreshbuffersolutionsandfollowinstructionsforusingapHmeter.
7. Autoclave,ifpossIBLe,at121ECfor20min.Somesolutionscannotbeautoclaved,forexample,SDS.Theseshouldbefiltersterilizedthrougha0.22μmfilter.Mediaforbacterialculturesmustbeautoclavedthesamedayitisprepared,preferablywithinanhourortwo.Storeatroomtemperatureandcheckforcontaminationpriortousebyholdingthebottleateyelevelandgentlyswirlingit
8. Solidmediaforbacterialplatescanbepreparedinadvance,autoclaved,andstoredinabottle.Whenneeded,theagarcanbemeltedinamicrowave,anyadditionalcomponents,e.g.antibiotics,canbeaddedandtheplatescanthenbepoured.
9. Concentratedsolutions,e.g.1MTris-HClpH=8.0,5MNaCl,canbeusedtomakeworkingstocksbyaddingautoclaveddouble-distilledwaterinasterilevesseltotheappropriateamountoftheconcentratedsolution.

Glassandplasticwareusedformolecularbiologymustbescrupulouslyclean.Dirtytesttubes,bacterialcontaminationandtracesofdetergentcaninhibitreactionsordegradenucleicacid.

Glasswareshouldberinsedwithdistilledwaterandautoclavedorbakedat150ECfor1hour.ForexperimentswithRNA,glasswareandsolutionsaretreatedwithdiethyl-pyrocarbonatetoinhibitRNaseswhichcanberesistanttoautoclaving.

Plasticwaresuchaspipetsandculturetubesareoftensuppliedsterile.Tubesmadeofpolypropyleneareturbidandareresistanttomanychemicals,likephenolandchloroform;polycarbonateorpolystyrenetubesareclearandnotresistanttomanychemicals.Makesurethatthetubesyouareusingareresistanttothechemicalsusedinyourexperiment.Micropipettipsandmicrofugetubesshouldbeautoclavedbeforeuse.

III.DisposalofBuffersandChemicals

1. Anyuncontaminated,solidifiedagaroragaroseshouldbediscardedinthetrash,notinthesink,andthebottlesrinsedwell.
2. Anymediathatbecomescontaminatedshouldbepromptlyautoclavedbeforediscardingit.PetridishesandotherbiologicalwasteshouldbediscardedinBiohazardcontainerswhichwillbeautoclavedpriortodisposal.
3. Organicreagents,e.g.phenol,shouldbeusedinafumehoodandallorganicwasteshouldbedisposedofinalabeledcontainer,notinthetrashorthesink.
4. Ethidiumbromideisamutagenicsubstancethatshouldbetreatedbeforedisposalandshouldbehandledonlywithgloves.Ethidiumbromideshouldbedisposedofinalabeledcontainer.
5. Dirtyglasswareshouldberinsed,alltracesofagarorothersubstancethatwillnotcomecleaninadishwashershouldberemoved,alllabelsshouldberemoved(ifpossible),andtheglasswareshouldbeplacedinthedirtydishbin.Bottlecaps,stirbarsandspatulasshouldnotbeplacedinthebinsbutshouldbewashedwithhotsoapywater,rinsedwellwithhotwater,andrinsedthreetimeswithdistilledwater.

IV.Equipment

A.GeneralComments

Itistoeveryone"sadvantagetokeeptheequipmentingoodworkingcondition.Asaruleofthumb,don"tuseanythingunlessyouhavebeeninstructedintheproperuse.Thisistruenotonlyforequipmentinthelabbutalsodepartmentalequipment.Reportanymalfunctionimmediately.Rinseoutallcentrifugerotorsafteruseandinparticularifanythingspills.Pleasedonotwastesupplies-useonlywhatyouneed.Ifthesupplyisrunninglow,pleasenotifyeithertheinstructorortheTAbeforethesupplyiscompletelyexhausted.Occasionally,itisnecessarytoborrowareagentorapieceofequipmentfromanotherlab.Exceptinanemergency,notifytheinstructor.

B.Micropipettors.

MostoftheexperimentsyouwillconductinthislaboratorywilldependonyourABIlitytoaccuratelymeasurevolumesofsolutionsusingmicropipettors.TheaccuracyofyourpipettingcanonlybeasaccurateasyourpipettorandseveralstepsshouldbetakentoinsurethatyourPipettesareaccurateandaremaintainedingoodworkingorder.Eachpairofstudentswillbeassignedasetofpipettorsanduponreceipt,theyshouldbelabeledwiththestudents"name.Theyshouldthenbecheckedforaccuracyfollowingtheinstructionsgivenbytheinstructor.Iftheyneedtoberecalibrated,doso.Wehavetwodifferenttypesofpipettors,RaininpipetmenandOxfordbenchmates.Sincethepipettorswillusedifferentpipettips,makesurethatthepipettipyouareusingisdesignedforyourpipettor.DONOTDROPITONTHEFLOOR.IfyousUSPectthatsomethingiswrongwithyourpipettor,firstcheckthecalibrationtoseeifyoursuspicionswerecorrect,thennotifytheinstructor.

C.UsingapHMeter.

BiologicalfunctionsareverysensitivetochangesinpHandhence,buffersareusedtostabilizethepH.ApHmeterisaninstrumentthatmeasuresthepotentialdifferencebetweenareferenceelectrodeandaglasselectrode,oftencombinedintoonecombinationelectrode.ThereferenceelectrodeisoftenAgCl₂.AnaccuratepHreADIngdependsonstandardization,thedegreeofstaticcharge,andthetemperatureofthesolution.

OperationofOrionPerpHecTpHMeter

1. Exposeholeonsideofelectrodebyslidingthecollardown.Makesurethereissufficientelectrodefillingsolutionintheelectrode(itshouldbeuptothehole).Ifnot,fillwithROSSfillingsolutiononly(Donotuseanyfillingsolutioncontainingsilver(Ag).
2. EnsurethatsampletobepHedisatroomtemperatureandisstirringgentlyonthestirplate.
3. CalibratethepHmeterwiththetwosolutionsthatbracketthetargetpH-4and7or7and10asfollows:
4. PresstheCALkeytoinitializethecalibrationsequence.Thelastcalibrationrangewillbedisplayed(e.g.7-4).PressYEStoacceptorusethescrollkeystoselectadifferentrange.PressYEStoaccept.
5. Thenumber7willlightuponthelefthandsideofthescreenindicatingthatthemeterisreadytoacceptthepH7standardbuffer.RinseoffelectrodeandplaceinfreshpH7standardbuffersolution.TheREADYlightwillcomeonwhenthevaluehasstabilized.PressYEStoacceptthevalue.
6. Thenumber4(or10)willlightupnextindicatingthatthemeterisreadytoacceptthepH4(or10)standardbuffersolution.RinseoffelectrodeandplaceinfreshpH4standardbuffersolution.TheREADYlightwillcomeonwhenthevaluehasstabilized.PressYEStoacceptthevalue.
7. SLPwillbedisplayed.ThemeterwillthengoMEASUREmode.
8. Rinseelectrodeandplaceintosample.TheREADYlightisdisplayedwhensignalisstable.

D.AutoclaveOperatingProcedures

Placeallmaterialtobeautoclavedinaautoclavabletray.Allitemsshouldhaveindicatortape.Separateliquidsfromsolidsandautoclaveseparately.Makesurelidsonallbottleareloose.Donotcrowdlargenumberofitemsintray-inorderforallitemstoreachtheappropriatetemperature,onemustallowsufficientair/steamcirculation.

1. Makesurechamberpressureisat0beforeopeningthedoor.
2. Placeitemstobeautoclavedintheautoclaveandclosethedoor.Some
3. autoclavesrequirethatyoualsolockthedoorafterit"sclosed.
4. Settime-typically20minutes.
5. Temperatureshouldbesetat121ECalready,butdouble-checkandchangeifnecessary.
6. Setcycle:Ifliquid,set"liquidcycle"or"slowexhaust".Ifdry,set"drycycle"or"fastexhaust"+drytime.
7. Startthecycle.Onsomeautoclaves,thecyclestartsautomaticallyatstep5.Onothers,turnto"sterilize".
8. Attheendofthecycle,checkthat:
   a.thechamberpressureisat0
   b.thetempis<100EC
9. Opendoor.(On3rdfloorautoclave,don"tpushendcycle)
10. Removecontentsusingglovesandimmediatelytightenallcaps.

E.OperatingInstructionsforSpectrophotometer-PharmaciaUltraspec

	Tomeasuretheabsorbanceofasolutionintheshort-waverange(<300nM)usethequartzcuvettes.
	Turnthespectrophotometeron-theswitchisontherightintheback.
	Allowtheinstrumenttocalibrate.Donotopenthechamberduringthistime.ThedeuteriumlampisOFFbydefault.ToreadabsorbanceintheUVrange,turnthedeuteriumlamponasfollowsafterthemachinehascompleteditscalibration:DepressthefunctionkeyuntilFn5isdisplayed.Pressthemodekeyuntild2onisdisplayed.Pressenter.Forbestaccuracy,thedeuteriumlampshouldbewarmedupfor20minutes.
	PressthefunctionkeyuntilFn0isdisplayed.Pressenter.Usingtheupordownarrowkeys,enterinthedesiredwavelength.
	Prepareareferencecuvettecontainingthesamediluentasyoursample.Prepareyoursample.
	Placethereferencecuvetteincell#1andplaceyoursamplesincells#2-6.
	Pressthecellkeyuntilcell#1isinposition.PresstheSetReferencekeytoblankagainsttheappropriatebuffer.Pressthecellkeytoadvancetoreadthenextsample.

V.WorkingwithDNA

A.Storage.

ThefollowingpropertiesofreagentsandconditionsareimportantconsiderationsinprocessingandstoringDNAandRNA.Heavymetalspromotephosphodiesterbreakage.EDTAisanexcellentheavymetalchelator.Freeradicalsareformedfromchemicalbreakdownandradiationandtheycausephosphodiesterbreakage.UVlightat260nmcausesavarietyoflesions,includingthyminedimersandcrosslinks.Biologicalactivityisrapidlylost.320nmirradiationcanalsocausecrosslinks,butlessefficiently.EthidiumbromidecausesphotooxidationofDNAwithvisiblelightandmolecularoxygen.Oxidationproductscancausephosphodiesterbreakage.Ifnoheavymetalarepresent,ethanoldoesnotdamageDNA.Nucleasesarefoundonhumanskin;therefore,avoiddirectorindirectcontactbetweennucleicacidsandfingers.MostDNasesarenotverystable;however,manyRNasesareverystableandcanadsorbtoglassorplasticandremainactive.5ECisoneofthebestandsimplestconditionsforstoringDNA.-20EC:thistemperaturecausesextensivesingleanddoublestrandbreaks.-70ECisprobableexcellentforlong-termstorage.Forlong-termstorageofDNA,itisbesttostoreinhighsalt(>1M)inthepresenceofhighEDTA(>10mM)atpH8.5.StorageofDNAinbuoyantCsClwithethidiumbromideinthedarkat5ECisexcellent.Thereisaboutonephosphodiesterbreakper200kbofDNAperyear.StorageofλDNAinthephageisbetterthanstoringthepureDNA.[Davis,R.W.,D.BotsteinandJ.R.Roth,AManualforGeneticEngineering:AdvancedBacterialGenetics.ColdSpringHarborLaboratories,ColdSpringHarbor,N.Y.1980.]

B.Purification.

Toremoveproteinfromnucleicacidsolutions:

1.treatwithproteolyticenzyme,e.g.,pronase,proteinaseK

2.PhenolExtract.ThesimplestmethodforpurifyingDNAistoextractwithphenolorphenol:chloroformandthenchloroform.Thephenoldenaturesproteinsandthefinalextractionwithchloroformremovestracesofphenol.

3.CsCl/ethidiumbromidedensitygradient

C.Quantitation.

1.Spectrophotometric.ForpuresolutionsofDNA,thesimplestmethodofquantitationisreadingtheabsorbanceat260nmwhereanODof1ina1cmpathlength=50μg/mlfordouble-strandedDNA,40μg/mlforsingle-strandedDNAandRNAand20-33μg/mlforoligonucleotides.Anabsorbanceratioof260nmand280nmgivesanestimateofthepurityofthesolution.PureDNAandRNAsolutionshaveOD₂₆₀/OD₂₈₀valuesof1.8and2.0,respectively.ThismethodisnotusefulforsmallquantitiesofDNAorRNA(<1μg/ml).

2.Ethidiumbromidefluorescence.TheamountofDNAisasolutionisproportionaltothefluorescenceemittedbyethidiumbromideinthatsolution.DilutionsofanunknownDNAinthepresenceof2μg/mlethidiumbromidearecomparedtodilutionsofaknownamountofastandardDNAsolutionsspottedonanagarosegelorSaranWraporelectrophoresedinanagarosegel.

D.Concentration.

Precipitationwithethanol.DNAandRNAsolutionsareconcentratedwithethanolasfollows:ThevolumeofDNAismeasuredandthemonovalentcationconcentrationisadjusted.Thefinalconcentrationshouldbe2-2.5Mforammoniumacetate,0.3Mforsodiumacetate,0.2Mforsodiumchlorideand0.8Mforlithiumchloride.TheionusedoftendependsonthevolumeofDNAandonthesubsequentmanipulations;forexample,sodiumacetateinhibitsKlenow,ammoniumionsinhibitT4polynucleotidekinase,andchlorideionsinhibitRNA-dependentDNApolymerases.TheadditionofMgCl₂toafinalconcentrationof10mMassistsintheprecipitationofsmallDNAfragmentsandoligonucleotides.Followingadditionofthemonovalentcations,2-2.5volumesofethanolareadded,mixedwell,andstoredoniceorat-20ECfor20minto1hour.TheDNAisrecoveredbycentrifugationinamicrofugefor10min(roomtemperatureisokay).ThesupernatantiscarefullydecantedmakingcertainthattheDNApellet,ifvisible,isnotdiscarded(oftenthepelletisnotvisibleuntilitisdry).Toremovesalts,thepelletiswashedwith0.5-1.0mlof70%ethanol,spunagain,thesupernatantdecanted,andthepelletdried.Ammoniumacetateisverysolubleinethanolandiseffectivelyremovedbya70%wash.Sodiumacetateandsodiumchloridearelesseffectivelyremoved.Forfastdrying,thepelletcanspunbrieflyinaSpeedvac,althoughthemethodisnotrecommendedformanyDNApreparationsasDNAthathasbeenoverdriedisdifficulttoresuspendandalsotendstodenaturesmallfragmentsofDNA.IsopropanolisalsousedtoprecipitateDNAbutittendstocoprecipitatesaltsandishardertoevaporatesinceitislessvolatile.However,lessisopropanolisrequiredthanethanoltoprecipitateDNAanditissometimesusedwhenvolumesmustbekepttoaminimum,e.g.,inlargescaleplasmidpreps.

E.RestrictionEnzymes

RestrictionandDNAmodifyingenzymesarestoredat-20ECinanon-frostfreefreezer,typicallyin50%glycerol.Theenzymesarestoredinaninsulatedcoolerwhichwillkeeptheenzymesat-20ECforsomeperiodoftime.Thetubesshouldneverbeallowedtoreachroomtemperatureandglovesshouldbewornwhenhandlingasfingerscontainnucleases.Alwaysuseanew,sterilepipettipeverytimeyouusearestrictionenzyme.Also,thevolumeoftheenzymeshouldbelessthan1/10ofthefinalvolumeofthereactionmixture.

VI.SterileTechnique

1.Allmedia,includingplates,liquidmediaandtopagarmustbeautoclavedimmediatelyafteritisprepared.Itisbesttopreparemediainseveralsmallbottles,onlyopeningoneatatime.Checkthebottleforcontaminationbeforeyouuseitbygentlyswirlingitandlookingforcloudymaterialinthecenter.Alwaysgrowupasmallamountofbrothalonewhengrowingcellsovernight.Asmallamountofcontaminationisnotalwaysevidentuntilthemediaisincubatedat37EC.

2.Useaflameoninoculatingloopsandonthelipsofmediabottlesbeforeandafterpipettingfromthem.Neverleaveamediaoragarbottleopenonthebenchanddon"stakeanindividually-wrappedpipetoutofitsprotectivewrapperuntilyouarereadytouseit(i.e.,don"twalkacrosstheroomwithanunwrappedpipet).Alwaysuseafresh,sterilepipetorpipettipwhenpipettingculturemedia,andnevergobackintoamediabottleorcellculturewithausedpipet.

3.Topreventwide-scale,untraceablecontamination,eachpersonshouldhavehisownstockofliquidculturemedia,topagar,plates,100%glycerol,glycerolstocksofcells,etc.anddon"tshare.

4.Overnightculturesshouldbegrownonlyfromasinglecolonyonafreshplateorfromapreviously-testedglycerolstockthatwasgrownfromasinglecolony.Toprepareanovernightculturefromaglycerolstock,takeanindividually-wrapped1-mlpipetandaculturetubeofmediatothe-80ECfreezer.Quicklyremovethecapfromthefreezervialcontainingtheglycerolstock,scrapasmallamountoficefromthesurfaceoftheculture,replacethecaponthefreezervial,andplacethepipetintotheculturetube.Sufficientnumbersofbacteriaarepresentintheiceinorderfortheculturetogrowtosaturationin16hours.Neverlettheglycerolstockthaw.

4.Thinkaboutwhatyouaredoing.Thebestdefenseiscommonsense.

VII.WorkingwithE.coli

A.SmallScaleCultures

ExperimentsusingE.colicellsshouldalwaysbedoneonfreshcultures,eitherfromafreshlystreakedplateorfromaglycerolstock.TogrowasmallscaleE.coliculture,prepare3-5mlofLB(orappropriatebroth-includeantibioticiftheculturecontainsaplasmid)intwosterile50mltubes.(Note:smallertubescanbeusedbuttheculturewillnotbeappropriatelyaeratedandhencewillnotgrowwellandisnotrecommended).Inoculateonetubewithasinglecolonyfromafreshplateorascrapingfromaglycerolstock.Thesecondtubeisusedasabrothcontrol.Incubatebothtubesat37EC,shakingvigorouslyovernight.Inspectthetubesthenextmorning.Thebrothcontrolshouldbeclearandtheinoculatedcultureshouldbeveryturbid.Makeanoteofanydebrisfoundinthetubesandonlyincubatelongerifthecultureisnotdense.Donotallowcellstoovergrow.Useimmediately.Forsomeapplications,cellscanbestoredat4ECforshortperiodspriortouse.

B.PermanentStorage

Foreverycultureused,inparticular,fornewlyconstructedstrainsorforcellscontainingplasmids,apermanentglycerolstockmustbepreparedassoonastheconstructhasbeenconfirmedandthisstockmustbeplacedinthelaboratorystockcollectionwiththeappropriatedocumentationandlocationinformation.Failuretofollowtheseprocedureswillresultinseriouspenalties.ThisprocedurepertainsnotonlytoE.colibuttoanyorganismforwhichadeepfreezestockcanbeprepared.Also,allplasmidconstructs,includingconstructionintermediates,mustbemaintainedincells,notasnakedDNAstocks.Foreachconstruct,atleast2stocksshouldbemade.ToprepareaglycerolstockforE.colicells,combine1.4mlofafreshlygrownovernightculturewith0.6mlofsterile50%glycerol.Mixwell.Transfertotwofreezervialslabeledwiththestrainname,thedateandyourinitials(notanEppendorftube).Immediatelyplaceintoadryice/ethanolbathorintoaboxinthe-80ECfreezer.Notethelocationandenterdataintothestrainbook.