Difference between revisions of "20.109(S09):Bacterial amplification of DNA (Day3)"
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==Introduction== | ==Introduction== | ||
− | [[Image:Be109transformation.jpg|thumb|right| | + | [[Image:Be109transformation.jpg|thumb|right|225px|'''Bacterial transformation''']] |
− | Assuming all went well, your reaction tubes from last time contain mutagenized DNA that encodes mutant inverse pericam. However, the desired DNA plasmid is likely present at a low concentration, and moreover it is | + | Assuming all went well, your reaction tubes from last time contain mutagenized DNA that encodes mutant inverse pericam. However, the desired DNA plasmid is likely present at a low concentration, and moreover it is nicked rather than in intact circular form. What we would like to do now is repair and further amplify only the mutagenized product. Thankfully, we have ''E. coli'' bacteria to do this for us quite efficiently! |
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+ | Bacteria can take up foreign DNA in a process called transformation, during which a single plasmid enters a bacterium and, once inside, replicates and expresses the genes it encodes. Most bacteria do not exist in a transformation-ready state, but can be made permeable to foreign DNA by chemical treatment or other means. Cells that are capable of transformation are referred to as competent. Competent cells are extremely fragile and should be handled gently, i.e., kept cold and not vortexed. Bacterial transformation is efficient enough for most lab purposes, resulting in as many as 10<sup>9</sup> transformed cells per microgram of DNA, but even with highly competent cells only 1 DNA molecule in about 10,000 is successfully transformed. Thus we need a way to identify transformed cells, which is usually accomplished with antiobiotics. For example, the plasmid carrying inverse pericam (calle [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=615 pRSET]) also carries a gene that leads to ampicillin-resistance. Consequently, a transformed bacterium will grow on ampicillin-containing agar medium, while untransformed cells will die before they can form a colony (see figure above right). Given the low concentration and nicked structure of your DNA to begin with, you should perform your transformations today with great care. | ||
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Before setting up transformations, you will test your mutagenized DNA for the presence and approximate concentration of product using gel electrophoresis. This technique separates large molecules by size using an applied electrical field and appropriate sieving matrix. DNA fragments are typically separated in gels composed of agarose, a seaweed-derived polymer (see figure, left). To prepare these gels, molten agarose is poured into a horizontal casting tray containing a comb. Once the agarose has solidified, the comb is removed, leaving wells into which the DNA sample can be loaded. The loaded DNA samples are then pulled through the matrix when a current is applied across it. Specifically, DNA molecules are negatively charged due to their phosphate backbones, and thus travel toward the positive charge at the far end of the gel (see figure, right). | Before setting up transformations, you will test your mutagenized DNA for the presence and approximate concentration of product using gel electrophoresis. This technique separates large molecules by size using an applied electrical field and appropriate sieving matrix. DNA fragments are typically separated in gels composed of agarose, a seaweed-derived polymer (see figure, left). To prepare these gels, molten agarose is poured into a horizontal casting tray containing a comb. Once the agarose has solidified, the comb is removed, leaving wells into which the DNA sample can be loaded. The loaded DNA samples are then pulled through the matrix when a current is applied across it. Specifically, DNA molecules are negatively charged due to their phosphate backbones, and thus travel toward the positive charge at the far end of the gel (see figure, right). | ||
− | [[Image:Be109agarosegelEM.jpg|thumb| | + | [[Image:Be109agarosegelEM.jpg|thumb|250px|left| Scanning EM image of agarose polymer]] |
− | [[Image:Be109agarosegelsetup.jpg|thumb|center| | + | [[Image:Be109agarosegelsetup.jpg|thumb|center|500px]] |
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<br style="clear:both;"/> | <br style="clear:both;"/> | ||
− | Although all DNA molecules travel in the same direction during gel electrophoresis, they do so at different rates: larger molecules get entwined in the matrix and retarded, while smaller molecules wind through the matrix more | + | Although all DNA molecules travel in the same direction during gel electrophoresis, they do so at different rates: larger molecules get entwined in the matrix and retarded, while smaller molecules wind through the matrix more quickly and thus travel further from the well. Ultimately, fragments of similar length accumulate into |