研究生分子生物学实验英文.docx

研究生分子生物学实验英文.docx

《研究生分子生物学实验英文.docx》由会员分享，可在线阅读，更多相关《研究生分子生物学实验英文.docx（49页珍藏版）》请在冰豆网上搜索。

研究生分子生物学实验英文

ExperimentalMethods

inMolecularBiology

SchoolofLifeScience

AnhuiUniversity

December，2005

Contents

DirectionalCloningintoPlasmidVectors.....................................................3

1.PreparationofPlasmidDNAbyAlkalineLysiswithSDS：

Midipreparation........................................................................................4

2.QuantitationofDNAandRNA...............................................................10

3.DigestingDNAWithRestrictionEnzymes.............................................12

4.GelElectrophoresisofDNA....................................................................15

5.InVitroAmplificationofDNAbyPCR..................................................20

6.IsolationofDNAFragmentsfromAgaroseGel......................................26

7.FreshCompetentE.ColiPreparedUsingCalciumChloride...................27

8.LigationReaction.....................................................................................28

9.TransformationofRecombinant...............................................................30

10.ExtractionofTotalDNAfromPlantTissue..........................................33

11.FastProteinLiquidChromatography（FPLC）…………...................….35

DirectionalCloningintoPlasmidVectors

1．PreparationofPlasmidDNAbyAlkalineLysiswithSDS：

Midipreparation

Plasmidsasvectors

Plasmidsaresmall，extrachromosomalcircularmo1ecules，from2toaround200kbinsize，whichexistinmultiplecopies（uptoafewhundred）wimhinthehostE.colicell。

Theycontainanoriginofreplication（ori），whichenablesthemtobereplicatedindependently，althoughthisnormallyreliesonpolymerasesandothercomponentsofthehostcell’smachinery．Theyusuallycarryafewgenes，oneofwhichmayconferresistancetoantibacterialsubstances．Themostwidelyknownresistancegeneisthebla，oramp’gene，encodingtheenzymeB-1actamase，whichdegradespenicillinantibioticssuchasampicillin．

Plasmidminipreparation

Theflrststepinasubcloningprocedure，forexamplethetransferofagenefromoneplasmidtonother，istheisolationofplasmidDNA．SinceplasmidsaresomuchsmallerthanE.colichromosomalDNA，theycanbeseparatedfromthelatterbyphysico-chemicalmethods，suchasalkalinelysis．ItisnormallypossibletoisolatesufficientplasmidDNAforinitialmanipulationfromafewmillilitersofbacterialculture．suchanisolationisnormallyknownasaminipreparationorminiprep．AsampleofanE.colistrainharboringtherequiredplasmidisinoculatedintoafewmilliltersofculturebroth．Aftergrowthtostationaryphase（overnight），thesuspensioniscentrifugedtoyieldacellpellet．

ThemostcommonlyusedmethodforthepurificationofplasmidDNAawayfromchromosomalDNAandmostoftheothercellconstituentsiscalledalkalinelysis．Thecellpelletisresuspendedinabuffersolutionwhichmayoptionallycontainlysozymetodigestthecellwallofthebacteria.Thecelllysissolution,whichcontainsthedetergentsodiumdeodecylsulfate（SDS）disruptsthecellmembraneanddenaturestheproteins;thealkalineconditionsdenaturetheDNAandbeginthehydrolysisofRNA．Thepreparationisthenneutralizedwithaconcentratedsolutionofpotassiumacetate（KOAc）atpH5．Thishastheeffectofprecipitatingthedenaturedproteins，alongwiththechromosomalDNAandmostofthedetergent（potassiumdodecylsulfateisinsolubleinwater）．Thesampleiscentrifugedagain，andtheresultingsupernatant（thelysate）nowcontainsplasmidDNA，which，beingsmallandclosed—circular，iseasilyrenaturedafterthealkalitreatment，alongwitha1otofsmallRNAmoleculesandsomeprotein．TherearenowinnumerableproprietarymethodsfortheisolationofpureplasmidDNAfromthe1ysateabove，manyofwhichinvolvetheselectivebindingofDNAtoaresinormembraneandthewashingawayofproteinandRNA．Theclassicalmethod，whichisslowerbutperfectlyeffective，involvesthephenolextractionofthelysatewithphenolorapheno1-chloroformmixture．Thephenolisimmisciblewiththeaqueous1ayerbutwhenmixedvigorouslywithit，thenallowedtoseparate，denaturestheremainingproteins，whichformaprecipitateattheinterfacebetweenthe1ayers．

TheDNAandRNAremainingintheaqueouslayerarenowconcentratedbyethanolprecipitation．Thisisageneralprocedure，whichmaybeusedwithanynucleicacidso1ution．IfsodiumacetateisaddedtothesolutionuntiltheNa+concentrationismorethan0.3M，theDNAand／orRNAmaybeprecipitatedbytheadditionof2-3volumesofethano1．Centrifugationwillpelletthenucleicacid，whichmaythenberesuspendedinasmallervolume，orinabufferwithnewconstituents，etc．Inthecaseofaminipreparation，ethanolisaddeddirectlytothephenol—extractedlysate，andthepelletistakenupinTris-EDTAsolution，thenormalsolutionforthestorageofDNA．ThissolutioncontainsTris-hydrochloridetobufferthesolution（usuallypH8）anda1owconcentrationofEDTA，whichchelatesandMg2+ionsinthesolution，protectingtheDNAagainstdegradationbynucleases，mostofwhichrequiremagnesium．RibonucleaseA（RNaseA）mayalsobeaddedtotheso1utiontodigestawayanyremainingRNAcontamination．ThisenzymedigestsRNAbutleavesDNAuntouchedanddoesnotrequireMg2+．

PreparationofCells

（1）Inoculate10mlofrichmedium（LB,YT,orTerrificBroth）containingtheappropriateantibiotic（12ulampicillinstooksolution）withasinglecolonyoftransformedbacteria（containingpUC118）．Incubatethecultureovernightat37℃withvigorousshaking．

LBmldlum（Luria-BertantMedium）：

Perliter：

To950mlofdeionizedH20，add：

bacto-tryptone10g

bacto—yeastextract5g

NaCl10g

Shakeuntilthesoluteshavedissoved．AdjustthepHto7．0with5NNaOH（-0.2m1）．AdjustthevolumeofthesolutiontolliterwithdeionizedH20．Sterilizebyautoclavingfor20minutesatl5lb／sq．in．onliquidcycle．Justbeforeautoclaving，addoneofthefollowing：

Bacto-agaroragarose（forplates）l5/liter

Bacto-agaroragarose（fortopagar）7g/liter

Whenthemediumisremovedfromtheautoclave，swirlitgentlytodistributethemeltedagaroragaroseevenlythroughoutthesolution．Allowthemediumtocoolto50℃beforeaddingantibiotics．Toavoidproducingairbubbles，mixthemediumbyswirling．Platescanthenbepoureddirectlyfromtheflask；allowabout25-30mlofmediumper90-mmplate．

Whenthemediumhashardenedcompletely，inverttheplatesandstorethem4℃untilneeded．Theplatesshouldberemovedfromstoragel-2hoursbeforetheyareused．Iftheplatesarefresh，theywill“sweat”whenincubatedat37℃．Thisallowsbacterialcoloniesorbacteriophageplaquestospreadacrossthesurfacesoftheplatesandincreasesthechancesofcross-contamination．Thisproblemcanbeavoidedbywipingoffanycondensationfromthelidsoftheplatesandthenincubatingtheplatesforseveralhoursat37℃inaninvertedpositionbeforetheyareused．

Ampicilin：

Stocksolution：

50mg/mlinH20；

Storage：

-20℃；

Workingconcentration：

60ug/ml

Stockso1utionsofampicillindissolvedinH20shouldbesterilizedbyfi1trationthrougha0.22-micronflter．

Toensurethatthecultureisadequatelyaerated：

①Thevolumeoftheculturetubeshouldbeatleastfourtimesgreaterthanthevolumeofthebacterialculture．

②Thetubeshouldbelooselycapped．

③Thecultureshouldbeincubatedwithvigorousagitation。

⑵Transferthecultureintoa1.5一mltubeandrecoverthebacteriabycentrifugationat12,000rpmfor30seconds．Removethemedium，leavingthebacterialpellet．

⑶Againtransferthecultureintoa1.5-mltubeandrecoverthebacteriabycentrifugationat12,000rpmfor30seconds．

⑷Removethemediumbygentleaspiration，leavingthebacterialpelletasdryaspossible．

ThepenaltyforfailingtoremovealltracesofmediumfromthebacterialpelletsisapreparationofplasmidDNAthatisresistanttocleavagebyrestrictionenzymes.Thisisbecausecell-wallcomponentsinthemediuminhibittheactionofmanyrestrictionenzymes.Thisproblemcanbeavoidedbyresuspendingthebacterialpelletinice-coldSTEandcentrifugingagain.

STE（alsocalledTEN）：

O.1MNaCl

l0mMTris-Cl（pH8.O）

1mMEDTA（pH8.O）

LysisofCells

（5）Resuspendthebacterialpelletin100ulofice-coldAlkalinelysissolutionIbyvigorousvortexingandstandthesuspensionatroomtemperaturefor5minutes．

solutionI：

50mMglucose

25mMTris-HCl（pH8.0）

10mMEDTA（pH8.0）

SolutionIcanbepreparedinbatchesofapproximatelyl00m1，autoclavedfor15minutesat1Olb／sq.in．onliquidcycle，andstoredat4℃．

MakesurethatthebacterialpelletiscompletelydispersedinAlkalinelysissolutionI．Vortexingtwomicrofugetubessimultaneouslywiththeirbasestouchingincreasestherateandefficiencvwithwhichthebacterialpelletsareresuspended．

Theoriginalprotocolcalledformeuseoflysozymeatthispointtoassistindissolutionofthebacterialcellwalls．Thisstepcanbesafelyomittedwhendealingwithbacterialculturesoflessthanl0mlinvolume.

（6）Add200u10ffreshlypreparedAlkalinelysissolutionIItoeachbacterialsuspension．Closethetubetightly,andmixthecontentsbyinvertingthetuberapidlyfivetimes．Donotvortex!

Storethetubeonicefor5minutes．

SolutionII：

0.2NNa0H（freshlydilutedfroma10Nstock）

1％SDS

MakesurethattheentireSurfaceofthetubecomesincontactwithAlkalinelysisso1utionII．Per10mlofsolutionII：

10NNa0H200ul

10％SDSlml

H2O8.8ml

（7）Add150ulofice-coldAlkalinelysissolutionIII．ClosethetubeanddisperseAlkalinelysisSolutionIIIthroughtheviscousbacteriallysatebyinvertingthetubeseveraltimes．Storethetubeonicefor3-5minutes．

SolutionⅢ：

5Mpotassiumacetate60ml