研究生分子生物学实验英文Word格式文档下载.docx
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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