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Dna Elektroforese Essay

1829 words - 7 pages

Chi Le og Thomas Rasmussen Svendborg Gymnasium Biologi Rapport Afleveres den 16/01-02
Biologi RapportomhandlendeDNA-elektroforeseUdarbejdet i samarbejde mellemChi Le, 2. BI/1Thomas Rasmussen, 2. BI/2Formål:Formålet med dette forsøg er først og fremmest at bestemme tilstedeværelsen af vores plasmid DNA, for derefter at bestemme størrelsen af dette ved hjælp af enDNA-eletroforese og autoradiografi.Teori:Først er det vigtigt at forstå princippet i DNA-elektroforese. Grundprincippet er, at man ved hjælp af strøm kan skille de forskellige molekyler af efter deres størrelse, form og ladning. Da DNA molekyler såvel som plasmider er negativt ladet pga. deres phosfat grupper i nukleotiderne, vil de alle vandre mod den positive pol. De er dog ikke alle lige store og da de mindste vil vandre hurtigst (og dermed længst) vil man på denne måde kunne adskille DNA'et efter antal baser i DNA molekylet. Det skal nævnes at forholdet mellem et DNA-stykkes størrelse og dets ladning er konstant så det bliver ikke mere negativt bare fordi det er større.Det skal dog her bemærkes at DNA-molekyle-strukturen også har en væsentligt indflydelse på hvordan molekylet vandre i mediet. En opremsning af vandringshastigheden med de hurtigste først vil se således ud: supercoilet, lineært, cirkulært og til sidst flere plasmider sammen.DNA bevæger sig ikke bare kontinuert i gelen. Det bevæger sig som en larve. Det trækker sig ind og strækker sig ud.For at DNA molekyler skal kunne bevæger sig skal de have et medium at gøre dette i. Dertil bruges et matrix for at hindre diffusion. Et matrix kan f.eks. være papir, bomuldsnor eller i dette tilfælde en agarosegel. Agarose er et lineært polysakkarid som produceres i bestemte rødalger. Denne agarosegel får sit netværk af porer pga. hydrogenbindinger mellem de lineære agarosepolymere. Jo større koncentrationen af agarose er jo tættere og mindre bliver porerne i gelen.Hvis koncentrationen af agarose blivefor høj vil DNA molekylerne ikke kunne vandre i gelen. Vi anvender en 0.7% agarosegel i hvilken vi vil kunne adskille DNA stykker i størrelses ordenen 0.8 - 10 kbp. Stykker på over 20 kbp vil være for store til at kunne adskilles. Det samme gælder stykker på under 0.1 kbp som vil vandre uden at støde på gelen og vil derfor ikke blive adskilt i forhold til hinanden.Strømstyrken har også betydning for elektroforesen. Hvis den er for høj smelter gelen. Jo lavere strømstyrke jo bedre adskillelse. Da vi har begrænset tid sætter vi strømstyrken til 30-35 mAmp, hvorved elektroforesen vil tage 1½ til 2 timer.For at kunne se resultaterne af vores DNA-elektroforese må vi farve...

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