pglo transformation lab ap lab 7 - brookings …：pglo转化实验室ap实验室7 -布鲁金斯…

《pglo transformation lab ap lab 7 - brookings …：pglo转化实验室ap实验室7 -布鲁金斯…》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。

1、pGLOTransformationLABAPLAB6http://www.mshri.on.ca/nagy/GFP%20mice.jpgBIO-RADlabbookpGLOoriblaGFParaCAequoreavictoria:Sourceof“glowinggene”forthisexperimentJellyfishGeneputintoOtherCrittershttp://www.technologyreview.com/files/21291/monkey_x600.jpghttp://www.lafuga.de/GFP_pig.jpgPLASMIDExtrachrom

2、osomalDNAOftencarrygenesforantibioticresistanceCanbepassedfromonebacteriumtoanotherhttp://www.agen.ufl.edu/~owens/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpgBacterialTransformationPlasmidsChromosomalDNABacterialCellTheuptakeofDNApGLOLABSUPPLIESFOAMtubeholder/flo

3、at4-fliptopmicrotubesBlue-Transformingsolution(CaCl2)Yellow-LBnutrientbrothPink-label+Purple-label-1-colorederaser(toIDyourtubesinwaterbath)1-pkgyellowinnoculatingloops2-Sterilepipettes4pouredagarplates1-LB2-LB/amp1-LB/amp/araPERMANENTMARKERCupwithcrushediceLABELTUBESPurple=+pGLOpink=-pGLOUsester

4、ilepipettetoadd250µLtransformationsolutiontopGLO+and–tubesTransformationsolution(CaCl2)GetyourrackonICE!INNOCULATETUBESWITHE.coliBACTERIAPickonecolonyTwirlloopin+pGLOtubeGetnewloopPickonecolonyTwirlloopin–pGLOtubeUSESPECIALGARBAGEBAGFORDISPOSALOFUSEDLOOPSEXAMINEpGLOplasmidDNAUseUVlighttoexa

5、minepGLOplasmidvialUVlightcanbeharmfultoyoureyes!Wearyourgoggles.Donotshineineyes.GFP=GreenFluorescentProteinisolatedfromjellyfishUSEDASAGENETICTOOLhttp://www.mshri.on.ca/nagy/GFP%20mice.jpgPLASMIDDNATRANSFERTHISSTEPISCRUCIAL!Lookcloselytomakesureyouhaveafilmofsolutionacrossthering.(Similartoso

6、apyfilmwhenyoublowbubbles)ADDPLASMIDTO+TUBEDONOTADDPLASMIDTO-TUBEPutrackonICEfor10MIN!WHILEYOURTUBESCOOLLABELYOURPLATESFLIPUPSIDEDOWNANDWRITELABELSONBOTTOM…NOTONTOP!LB(LuriaandBertani)–broth&agarprovidesnutrientsforbacterialgrowthLB/ampLuriaagar+ampicillin(antibiotic)LB/amp/araLuriaagar+ampicill

7、in+arabinosesugarSHOCKINGINCREASESUPTAKEOFFOREIGNDNA(PLASMID)OSMOTICSHOCK=TransformingsolutionCaCl2HEATSHOCKRAPIDTEMPERATURECHANGEisthekey50SECONDS2MINUTESPlacefoamrackwith+and–tubesondesktopUsenewsterilepipettetoadd25