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SectionG:Genemanipulation华中师范大学生科院杨旭分子生物学研究方法分子生物学研究的内容生物大分子结构和功能：A,B,C,D,E,F分子生物学研究方法：G,H,I,J基因表达与调控：L,M,N,O,P,Q其他(病毒基因组等)：R,S分子生物学研究方法包括：SectionG:GeneManipulation(基因操作技术)SectionH:CloningVectors(克隆载体)SectionI:GeneLibraries(基因文库)SectionJ:AnalysisofclonedDNA(克隆DNA的分析)2014-03-25SectionG:Genemanipulation华中师范大学生科院杨旭SectionG

GeneManipulation(基因操作技术)

G1DNAcloning:anoverview

G2PreparationofplasmidDNA

G3Restrictionenzymesandelectrophoresis

G4-1Ligation

G4-2Transformation

G4-3AnalysisofbinantsSectionG:Genemanipulation华中师范大学生科院杨旭DNAcloning:基本步骤1.外源性DNA片段的获取2.DNA重组:外源DNA片段与载体分子的体外连接3.转化:将重组DNA导入它们能够复制的宿主细胞4.克隆基因的分离与鉴定:SubcloningTheMakingofLibraries文库载体功能载体基因应用GeneApplication以plasmid(vector)和E.coli(host)为例SectionG:Genemanipulation华中师范大学生科院杨旭G1DNACloning:AnOverview

(DNA克隆概述)DNAcloning(DNA克隆)Sub-cloning(亚克隆)DNAlibraries(DNA文库)Screeninglibraries(筛选文库)Vectors(载体)Plasmids(质粒)Hosts(宿主)Analysisofclone(克隆分析)SectionG:Genemanipulation华中师范大学生科院杨旭DNAcloning:（DNA克隆的定义）Definition:Itisthemakingprocesstoplacethetarget(目的)gene(thegeneofinterest)inavector(anautonomously(自主)replicatingpieceofDNA),formingbinantDNA,whichthenisplacedintoanotherhostspecies.DNA克隆：在分子生物学的研究中，人们（1）把含有目的基因的外源性DNA插入具有复制能力的载体DNA中，形成重组DNA；（2）再将这种重组DNA转化到宿主细胞中，形成“转化子”的操作技术，称为“DNA克隆”、“基因克隆”或简称“克隆”（“转化子”具有使目的基因得以永久保存、复制和扩增的能力）。EEStargetgeneHosttransformant抗生素抗性基因vectorEOri克隆：原意为“无性增殖”SectionG:Genemanipulation华中师范大学生科院杨旭DNAcloning:基本步骤1.外源性DNA片段的获取

(1)PreparationofplasmidDNAcontainingtheclonedtargetgene.(2)Digestionoftheplasmidwithrestrictionendonucleases.(3)Separationofthefragmentsbyagarosegelelectrophoresis.(4)Purificationofthedesiredtargetfragment.2.DNA重组:外源DNA片段与载体分子的体外连接

(5)Ligationofthefragments,toformanewbinantmolecule.3.转化:将重组DNA导入它们能够复制的宿主细胞

(6)TransformationoftheligatedplasmidintoanE.colistrain(株).4.转化子的分离与鉴定:(7)Selectionoftransformedbacteria(seeTopicG4).(8)Analysisofbinantplasmids(seeTopicG4).以plasmid(vector)和E.coli(host)为例SectionG:Genemanipulation华中师范大学生科院杨旭Definition:ItistheprocesstotransferofafragmentofclonedDNAfromonevectortoanother.Subcloning(亚克隆：从载体到载体)SubcloningTheMakingofLibraries文库载体功能载体工作载体基因应用GeneApplicationSectionG:Genemanipulation华中师范大学生科院杨旭Experimentalsteps:1.Preparation:oftheplasmid2.Digestionoftheplasmid3.Separationofthefragments4.Purificationoftargetfragment5.Ligationofthefragments6.Transformation

7.Selectionoftransformedbacteria.8.Analysisofplasmids.+-质粒分析Subcloning(亚克隆：从载体到载体)SectionG:Genemanipulation华中师范大学生科院杨旭DNAlibraries

(DNA文库)Definition:DNAlibrariesaresetsofDNAclones(vectors/hosts),eachofwhichhasbeenderivedfromtheinsertofadifferentfragment(targetgenes)intoavectorfollowedbypropagation(增殖)inthehost.Classificationandfeatures:1.Genomiclibraries2.cDNAlibraries获取外源性DNASectionG:Genemanipulation华中师范大学生科院杨旭1.GenomiclibrariesGenomiclibraries:TheyarederivedfromrandomfragmentsofDNAfromthegenomesof

speciesbyshotgunapproach(鸟枪法);Theapproachmaybeaninefficientoffindingagene,especiallyineukaryoticgenomes,wheremuchoftheDNAisnoncoding.SectionG:Genemanipulation华中师范大学生科院杨旭2.cDNAlibrariesProtein

Cell

mRNAcDNAVectorHostTheyarederivedfromthemRNAbyreversetranscriptionandaretheninsertedintoavector;cDNAlibrariesareefficientforfindingandcloningagene,butonlythecodingregion,notthesurroundinggenomicsequences.SectionG:Genemanipulation华中师范大学生科院杨旭Screeninglibraries(筛选文库:寻找目的基因)Definition:ItistheprocesstouseaDNAprobetofindthetransformantsthatcontaintargetgenes.DNAprobe:ItisaradioactivelylabeledshortDNAsequencewhichispartiallycomplementarytoaregionofthetargetgenesequence,therefor,thetargetgeneorclonecanbedetectedbyitshybridization.Makingofthe

probes:Theprobesequencemightbeanoligo-nucleotide(寡核苷酸)derivedfromthesequenceoftheproteinproductofthegene;Fromarelatedgenefromanotherspecies.Anincreasinglyimportantmethodforthegeneration(生产)ofprobesisPCRSectionG:Genemanipulation华中师范大学生科院杨旭Vectors-I(载体的定义和特征)定义:具有自主复制能力的DNA分子就是所谓的分子克隆的载体，质粒、噬菌体、病毒和人工染色体等小分子量的复制子都可以作为导入基因的载体。SectionH将专门讲解用于基因克隆的载体Thefeaturesofvectors:Vectorsmustnormallybecapableofbeingreplicatedandisolatedindependentlyofthehost'sgenome(Ori-C);Vectorsalsohaveaselectablemarker,agenewhichallowshostcellsconferringresistancetoatoxin.Therearesomevectors,forexamplephage

(seeTopicH2),whichcanincorporateDNAintothehostgenomeforlongertermexpressionofclonedgenes.

SectionG:Genemanipulation华中师范大学生科院杨旭Vectors-II(载体的种类)Thecommonvectors:1.Plasmids:CircularplasmidofE.coli:usedinE.coli(host);Yeastepisomal(游离型)plasmids:usedinyeast;Agrobacteriumtumefaciens(根瘤农杆菌)Tiplasmid:inplant.2.Bacteriophages(virusesinfectingbacteria;seeTopicR2):Phage:alsobeenusedinE.coli,forcloninglargerfragmentsPhageM13:usedtoclonessDNAusedinE.coli.3.Cosmids(粘粒plasmid-bacteriophagehybrids):(seeTopicH3).4.Artificialchromosomes:forcloninghugefragmentsfromhumans.BAC:Bacterialartificialchromosomes(inE.coli);YAC:Yeastartificialchromosomes(inYeast).5.Virus:forothereukaryoticcellsinculture(培养状态)SV40:Simianvirus40(猴空泡病毒40);Retroviruses.(seeTopicH4).SectionG:Genemanipulation华中师范大学生科院杨旭Plasmids(最常见的载体--质粒)Thefirstcloningvectorstobeused,inthemid1970s,werenaturalplasmidsoriginallyfromE.coli.Structureandfeatures:Plasmidsare

smallinsize,from2toaround200kb

extra-chromosomalcircularmolecules

whichexistinmultiplecopies(uptoafewhundreds)

withinthehostE.colicells.

Theycontainanoriginofreplication(ori),whichenablesthemtobereplicatedindependently.

SectionG:Genemanipulation华中师范大学生科院杨旭Plasmids(最常见的载体--质粒)Resistance

gene:Theyusuallycarryafewgenes,oneofwhichmayconfer(赋予)resistancetoantibacterialsubstances:1.Themostwidelyknownresistancegeneisamprgene,whichencodestheenzyme

-lactamase(-内酰胺酶),thatdegradespenicillinantibioticssuchasampicillin(氨苄青霉素).2.

AnotheristhetetAgene,whichencodesatransmembranepumpthatisabletoremovetheantibiotictetracycline(四环素)fromthecell.EOriamprSectionG:Genemanipulation华中师范大学生科院杨旭Hosts(宿主细胞的定义和种类)Host的定义：含有重组DNA分子的细胞，能够使目的基因得以保存、复制和扩增，包括原核细胞和真核细胞。单克隆的定义：带有某一种重组DNA的宿主细胞，增殖构成的一群具有遗传一致性的细胞。Thecommonhosts:E.coli

(大肠杆菌):TheinitialisolationandanalysisofDNAfragmentsisalmostalwayscarriedoutusingtheE.coliasthehost(forcircularplasmid,phage,phageM13,cosmid,BAC);Yeast

(酵母菌):Itisbeingusedtomanipulate(制备)verylargefragmentsofthehumangenome(forepisomalplasmid,YAC).Othercells，例如:根瘤农杆菌(forTiplamid)、昆虫培养细胞(forbaculovirus杆状病毒)、哺乳动物培养细胞(forSV40,retroviruses,shuttlevectors)、枯草杆菌、动物细胞等。SectionG:Genemanipulation华中师范大学生科院杨旭Analysisofclone(克隆分析)Onceaclonecontainingatargetgeneisidentified,thestructureoftheclonedfragmentmaybeinvestigated:Restrictionmapping(限制性酶切图谱)TheanalysisofthefragmentationoftheDNAwithrestrictionenzymes(seeTopicJ1)andbyagarosegelelectrophoresisusingmarkerofknownsizes.Sequencing(测序)oftheentirefragment(TopicJ2).DNAfunctionalanalysisThesequencecanthenbeanalyzedbycomparisonwithotherknownsequencesfromdatabases,andthecompletesequenceoftheproteinproductdetermined(seeTopicJ2).SectionG:Genemanipulation华中师范大学生科院杨旭一、外源性DNA片段的获取G2PreparationofPlasmidDNA(质粒的制备)

Plasmidminipreparation(质粒的小量制备)G3RestrictionEnzymesandElectrophoresis(Digestion,separationandpurification)Restrictionendonucleases(限制性内切核酸酶)Restrictionsequences(限制性识别序列)Cohesiveends(粘末端)Restrictiondigests(限制性酶解)Agarosegelelectrophoresis(琼脂糖凝胶电泳)SectionG:Genemanipulation华中师范大学生科院杨旭Plasmidmini-preparation

(质粒的小量制备)Bacterialculture(细菌培养)Alkalinelyses(碱裂解)Phenolextraction(酚抽提)Ethanolprecipition(乙醇沉淀)Cesiumchloridegradient(氯化铯梯度)SectionG:Genemanipulation华中师范大学生科院杨旭振动培养离心集菌细菌1.悬浮细胞沉淀;2.加入溶菌酶；3.加去污剂SDS;4.NaOH(碱裂解)质粒RNA蛋白质蛋白质、染色体DNA、膜变性蛋白酚--氯仿蛋白质染色体DNA质粒RNA质粒RNA水溶液

纯化质粒1.取水溶液2.乙醇沉淀3.用RNA酶1.取质粒带2.乙醇沉淀酚-氯仿抽提CsCl梯度离心质粒的小量制备流程SectionG:Genemanipulation华中师范大学生科院杨旭Digestion:Restrictionendonucleases

(限制性内切核酸酶)Function:Restriction-modificationsystemsoccurinmanybacterialspecies,andconstitute(构成)adefensemechanismagainsttheforeignDNAintothecell.Structure:Restriction-modificationsystemconsistoftwoparts：1.Thefirstpartisarestrictionendonuclease,whichrecognizesashort,symmetricalDNAsequence(Fig.1),andhydrolyzestheDNAbackboneineachstrandataspecificsite.2.Thesecondpartisamethylase,whichaddsamethylgrouptoaCorAbasewithinthesamerecognitionsequences.ThismodificationprotectsthehostDNAagainsttheendonuclease.SectionG:Genemanipulation华中师范大学生科院杨旭5’-GAATTC-3’3’-CTTAAG-5’Digestion:Restrictionsequences(识别序列)Definition:Itisashortpalindromic(回文)sequences,atwhichrestrictionenzymescleaveDNAsymmetrically(对称地)inbothstrands.

ActingSteps:EcoRIasanexample.Recognizing:TherestrictionendonucleasesEcoRIactsasadimer,willonlyrecognizea6bppalindromic(回文)sequence.Cutting:Theproductofthecuttingreactionistworestrictionfragments,eachwitha5'-endwithaphosphategroupanda3'-endwithafreehydroxylgroup.5’-G-OH

P-AATTC-3’3’-CTTAA-P

HO-G-5’5’-GAATTC-3’3’-CTTAAG-5’RecognizingCuttingannealingEcoRISectionG:Genemanipulation华中师范大学生科院杨旭Digestion:Cohesiveends(粘末端)Definition:Someoftheproductsofrestrictionenzymedigestionhaveprotruding(突出的)ends,andtheseendsareknownascohesive,or'sticky'ends.

Features:Thoseproductsofrestrictionenzymedigestionwithprotrudingendshaveafurtherproperty:

Theycanbindtoanyotherendwiththesameoverhanging(突出的)sequence,bybasepairing(annealing退火配对)ofthesingle-strandedtails.

Forexample,anyfragmentformedbyanEcoRIcutcanannealtoanyotherfragmentformedinthesameway,andmaysubsequentlybejoinedcovalentlybyligation.

Infact,insomecases,DNAendsformedbyenzymeswithdifferentrecognitionsequencesmaybecompatible(兼容)SectionG:Genemanipulation华中师范大学生科院杨旭Digestion:Restrictiondigests-I(限制性酶解)Application:DigestionofplasmidorgenomicDNAiscarriedoutwithrestrictionenzymesforanalyticalorcloningpreparationpurposes.Examples:Thedigestionofasampleplasmidwithtwodifferentrestrictionenzymes,BamHIandEcoRI.

PlasmidwithgeneXEEEEXEBBBBamHIEcoRIEEBSectionG:Genemanipulation华中师范大学生科院杨旭Digestion:Restrictiondigests-II(限制性酶解)Reactionsystem:AllrestrictionenzymesrequireMg2+(magnesium)usuallyataconcentrationofupto10mM;butdifferentenzymesrequiredifferent:

pHs,

NaClconcentrationsor

othersolutionconstituents(成份).Reactionprocess:TheDNAis

incubatedatthe

optimumtemperature(37C)with

theenzymeand

theappropriatebuffer,inavolumeofperhaps20l.

Adyemixtureisthenaddedtosolution,and

thesampleisloaded(上样)ontoanagarosegel.SectionG:Genemanipulation华中师范大学生科院杨旭Separation:Agarosegelelectrophoresis-I

(琼脂糖凝胶电泳)Definition:Agaroseisapolysaccharidederivedfromseaweed,whichformsasolidgel(固体凝胶)whendissolvedinaqueoussolutionatconcentrationsbetween0.5and2%(w/v).

Principle(原理):Movement:Whenanelectricfieldisappliedtoanagarosegelinthepresenceofabuffersolution,DNAfragmentsmovethroughthegeltowardsthe+electrode,becauseDNAnegativelycharged.Movementrate:ThemovementrateisdependentonfragmentsizesandshapesoftheDNAandtheelectrophoresismaybeusedtoseparatemixturesofDNAfragmentsbytherate.+-DNAsampleAgarosegelMigrationofDNA电导缓冲液SectionG:Genemanipulation华中师范大学生科院杨旭Generalprocedures:TheDNAsamplesareplacedinwells(上样孔)inthegelsurface.thepowersupplyisswitchedonandtheDNAisallowedtomigrate(迁移)throughthegelinseparatelanesortracks(泳道).Theaddeddye(EB)alsomigrates,andisusedtofindtheprogress.TheDNAisstainedbytheEBinthegel,theDNAshowsupasanorangebandonillumination(光照)byUVlight.Separation:Agarosegelelectrophoresis-II

(琼脂糖凝胶电泳)SectionG:Genemanipulation华中师范大学生科院杨旭Resultreading:seep103Fig4TracksM:AsetoflinearmarkerDNAfragmentsofknownsizes;TrackU:UndigestedplasmidDNAconsistsoftwobands.ThelowerbandconsistsofnegativelysupercoiledplasmidDNA.Theupperbandisthenicked(缺口)DNA,whichhaslowermobility;TrackB:digestionwithBamHI,containingasinglefragment;TrackE:digestionwithEcoRI,containingfivefragments,thethirdlargestfragmentcontainsthegeneX,whichcouldbepurifiedfromthegelandreadyforligationintoanewvectorSeparation:Agarosegelelectrophoresis-III

(琼脂糖凝胶电泳)PlasmidwithgeneXEEEEXEBSectionG:Genemanipulation华中师范大学生科院杨旭Purificationoffragments(片段的纯化)Purposes:AgarosegelsmayalsobeusedpreoperativelytoisolatespecificfragmentsforuseinsubsequentligationorothercloningexperimentsPurificationmethod:Fragmentsarecutfromthegel,andtreatedbyoneofanumberofprocedurestopurifytheDNAawayfromthecontaminatingagarose.Example:IfweassumethattheEcoRIfragment,whichcontainsthegeneX,isthetargetDNAforasubcloningexperiment,thenthethirdlargestfragment(intrackEofFig.4a)couldbepurifiedfromthegel,readyforligationintoanewvector(seeG4).SectionG:Genemanipulation华中师范大学生科院杨旭+-质粒分析PurificationSectionG:Genemanipulation华中师范大学生科院杨旭二、DNA重组

外源DNA片段与载体分子的体外连接

G4-1Ligation(DNAbination)DNAligation(DNA连接)binantDNAmoleculesAlkalinephosphatase(碱性磷酸酶)上课SectionG:Genemanipulation华中师范大学生科院杨旭DNAligation(DNA连接)Purpose:ToinsertatargetDNAfragmentintoavector.FunctionofDNAligases:theywillrepair(ligate)abreakinonestrandofadsDNAmolecule.Energysources:DNAligasefromE.coliusesNAD+

asenergysource;T4DNAligaseusesATP(morecommonlyused).Differentends:Cohesiveends:Ligasesareefficientatsealingthebrokenphosphodiesterbondsforcohesiveends,Bluntends:justT4ligasecanevenligateonebluntendtoanother,butwithratherlowerefficiency.SectionG:Genemanipulation华中师范大学生科院杨旭binantDNAmolecules-I(重组DNA分子)MakingofthebinantDNA:TargetDNA:maybeasinglefragmentisolatedfromagarosegel,oramixtureofmanyfragmentsfromgenomicDNA.VectorDNA:thenewvectorDNAcanbecutwiththesameenzyme.IfthetargetDNAhasbeenpreparedbydigestionwithsameenzyme(EcoRI).binantmolecules:TheproductsarecircularmoleculeswiththetargetfragmentinsertedattheEcoRIsiteofthevectormoleculewitheitherorientation(方向).EEEcoRIEESEES抗生素抗性基因OriEAEBSectionG:Genemanipulation华中师范大学生科院杨旭binantDNAmolecules-II

(重组DNA分子)Aproblem:Therecreationoftheoriginalvectorplasmid,bycircularizationofthelinearvectoralone,isacompetingsidereactionwhichcanmakeproblemsonthebinantidentification.

Solutionone--Paireddistinctrestriction:OnesolutionistoprepareboththetargetDNAandthevectorDNAusingapairofdistinctrestrictionenzymes,suchthattheyhavepatiblecohesiveendsateitherend.Thelikelihood(可能性)ofligatingthevectorintoacircleisthenmuchreduced.SectionG:Genemanipulation华中师范大学生科院杨旭HO

-AATTCHO-GG-OHCTTAA-OHAlkalinephosphatase(碱性磷酸酶)Solutiontwo:Linearvector:Alkalinephosph-ataseremovesphosphategroupsfromthe5'-endsofDNAmolecules.Thelinearvectorwillhencebeunabletoligateintoacircle,sincenophosphatesareavailable.TargetDNA:AligationwithatargetDNAinsertcanstillproceed,sinceonephosphateispresenttoligateonestrandateachcutsite.ThenicksintheotherstrandswillberepairedwhentransformatedinE.coli.EEEESPPG-OHCTTAA-OHHO-AATTCHO-G碱性磷酸酶SectionG:Genemanipulation华中师范大学生科院杨旭三、转化:

将重组DNA导入它们能够复制的宿主细胞

G4-2Transformation(转化)Transformation(转化)Selectionofplates(平板选择培养法)Transformationefficiency(转化率)Growthandstorageoftransformants(转化子增殖和保存)SectionG:Genemanipulation华中师范大学生科院杨旭Transformation-I(转化)Definition:Itistheprocessoftake-up(吸收)offoreignDNA(normallyplasmids)bybacteria.Plasmidsarecloned(无性繁殖)bytransferringintoE.coliwithdefinedgeneticproperties.Purpose:Thebinantandotherplasmidformedbyligationmustbeisolatedfromoneanotherandreplicatedbyahost.Hosts:ThemostcommonhostsarestrainsofspecialE.coli,whichmustnotexpressarestriction-modificationsystem.Thehostcellcanacceptonlyonekindofforeignplasmid.Competentcells(感受态细胞):ItwasdiscoveredthatE.colicellstreatedwithsolutionscontainingCa2+ionswererendered(表达出)susceptible(易感性)totakeup(吸收)exogenousDNA.CellspretreatedwithCa2+,inordertorenderthemabletotakeupDNA,areknownascompetentcells(商品化的更好用).SectionG:Genemanipulation华中师范大学生科院杨旭Transformation-II(转化)Transformationprocess:TakingupDNA:Asolutionofaplasmidmoleculeiscombinedwithasuspension(悬浮液)ofcompetentcellsforaperiod(30-40min),toallowtheDNAtobetakenup.Recovering:Themixtureisthenheat-shocked(热激)at42ºCfor1-2min.ThisstepinducesenzymesinvolvedintherepairofDNA,whichallowthecellstorecoverfromunusualconditionsofthetransformationprocess,andincreasestheefficiency.Transformation:Thecellsarethenincubatedinagrowthmediumandfinallyspreadonanagarplateandincubateduntilsinglecoloniesofbacteriagrow.

plasmid感受态细胞TakingupDNARecoveringTransformationSectionG:Genemanipulation华中师范大学生科院杨旭Selectionofplates(平板选择培养法)Aproblem:Ifallthecompetentcellspresentinatransformationreactionwereallowedtogrowonanagarplate,thenmanythousandsormillionsofcolonieswouldresult.Hence,amethodfortheselectionofclonescontainingaplasmidisrequired.Principle:Thisisalmostalwaysprovidedbythepresenceofanantibioticresistancegeneontheplasmidvector,forexamplethe-lactamasegene(ampr)conferringresistancetoampicillin.Ifthetransformedcellsaregrownonplatescontainingampidllin,onlythosecellswhichareexpressing-lactamase(duetothepresenceofatransformedplasmid)willsurviveandgrow.Notice:Ifaligationmixture(butnotasingleplasmidmolecules)hadbeenusedforthetransformation,wewouldnotknowatthisstagewhichclonescontainbinantplasmidswithatargetfragmentincorporated.SectionG:Genemanipulation华中师范大学生科院杨旭Transformationefficiency(转化率)Definition:Thequalityofcompetentcellsmaybemeasuredbydeterminingthetransformationefficiency,definedasthenumberofcolonies(菌落数)formedper

gpureplasmids.Rangeandusage:Transformationefficienciescanrange:103colonies/

gplasmids:canbeusedforcrudetransformationprotocols,whichwouldonlybeappropriatefortransferringanintact(完整)plasmidtoanewhoststrain(subcloning);105colonies/

gplasmids:wouldbeenoughforasimplecloningexperimentofthekindoutlinedinthisbook.>108colonies/

gplasmids:canbeusedforgenerationoflibraries;

SectionG:Genemanipulation华中师范大学生科院杨旭GrowthandstorageoftransformantsGrowth:Method:Singlecoloniesfromatransformationplatearetrans-ferredtobrothandgrownovernightuntilthestationaryphase.Notice:Thebroth(培养液)mustbeincludedwiththeantibiotic,andusedtoselectthetransformantsontheoriginalplate,tomaintaintheselectionforthepresenceoftheplasmid.Storage:Method:Itisnormalpracticetoprepareastockofeachcultureatthisstage,byfreezingaportion(部分)ofthecultureinthepresenceofglycerol(甘油，或者二甲亚砜).Reason:Theeffectofglycerolstock(贮存液)istoprotectthecellsfromicecrystalformation.Thestockwillenablethesamestrain/plasmidtobegrownan