杨荣武分子生物学课件Week2modification

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1、DNAandRNAisolation,purification,visualizationandquantitationGenomicDNApreparationoverviewPlasmidDNApreparationDNApurificationPhenolextractionEthanolprecipitationRNAworkWhatdoweneedDNAfor?Detect,enumerate,clonegenesDetect,enumeratespeciesDetect/sequencespecificDNAregionsCreatenewDNA“construct

2、s”(recombinantDNA)WhataboutRNA?Whichgenesarebeingtranscribed?When/wherearegenesbeingtranscribed?Whatistheleveloftranscription?cellgrowthcellharvestandlysisDNApurificationDNApurification:overviewDNAconcentrationBacterialgenomicDNAprep:cellextractLysis:DetergentsOrganicsolventProteases(lysozym

3、e)Heat“cellextract”GenomicDNAprep:removingproteinsandRNAAddtheenzymeRNasetodegradeRNAintheaqueouslayerNeedtomixgently!(toavoidshearingbreakageofthegenomicDNA)chloroform2waystoconcentratethegenomicDNA70%finalconc.“spooling”EthanolprecipitationGenomicDNAprepinplants--howgetridofcarbohydrates?

4、CTAB:Cationicdetergent(MC6.61-6.62)(lowionicconditions)N+CH3Br-CH3CH3C16H33Plasmids:vehiclesofrecombinantDNABacterialcellgenomicDNAplasmidsNon-chromosomalDNAReplication:independentofthechromosomeManycopiespercellEasytoisolateEasytomanipulatePlasmidpurification:alkalinelysisAlkalineconditions

5、denatureDNANeutralize:genomicDNAcan’trenature(plasmidsCANbecausetheyneverfullyseparate)DNApurification:silicabindingBindingoccursinpresenceofhighsaltconcentration,andisdisruptedbyelutionwithwaterDNApurification:phenol/chloroformextraction1:1phenol:chloroformor25:24:1phenol:chloroform:isoamyl

6、alcoholPhenol:denaturesproteins,precipitatesformatinterfacebetweenaqueousandorganiclayerChloroform:increasesdensityoforganiclayerIsoamylalcohol:preventsfoamingAqueousvolume(atleast200microliters)Add2volumesofphenol:chloroform,mixwellSpinincentrifuge,moveaqueousphasetoanewtubeRepeatsteps2and3

7、untilthereisnoprecipitateatphaseinterface(extractaqueouslayerwith2volumesofchloroform)PhenolextractionEthanoldepletesthehydrationshellsurroundingDNA…AllowingcationstointeractwiththeDNAphosphatesReducingrepulsiveforcesbetweenDNAstrandsCausingaggrega