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BISC219/F12: RNAi Lab 7: Difference between revisions
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→Picking a Bacterial Colony
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This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. Because bacteria reproduce asexually and are immobile on solid media, it is likely that the hundreds of thousands of bacteria making up that colony are genetically identical daughters of a single cell. This allows us to take bacteria from a single colony and sub-culture them in liquid media to make millions of identical copies. However, knowing that the bacteria growing in your broth or on your agar with ampicillin all have the plasmid responsible for amp resistance does not confirm that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that may have a plasmid lacking the gene of interest. That can happen when a vector plasmid is "empty". Our plasmid, pPD129.36 ''lsy-2'', was genetically modified from a purchased base plasmid genetically synthesized by a pharmaceutical company. Remember that researchers need to be able to study any gene of interest; therefore, base plasmids are created that allow insertion of any gene of interest in proper alignment with synthetic gene promoters so that the gene of interest can be expressed as desired in a bacterial or a yeast model system. The first step in this process after acquiring a base plasmid (such as pPD129.36) is to isolate small segments of chromosomal DNA that contain your gene. Then you must make lots of copies of your gene by PCR after designing primers that will copy it. The next step is to ligate (insert) the gene from the PCR product into the vector plasmid so that it is in proper alignment with the promoters. If all goes well the ligation works and you achieve this proper alignment and after transformation (uptake of the plasmid by a cell). both the selection gene and the gene of interest are expressed. However, to do a ligation you must cut the vector plasmid at specific places in the DNA using specific restriction enzymes and then reanneal the ends after the gene of interest is inserted. Occasionally that ligation/annealing process fails to work properly and the plasmid DNA anneals back on itself. We need a way to find only the colonies with successful ligation that are expressing our ''C. elegans lys-2 gene'' off the synthetic vector plasmid.<br> | This process of using a marker (usually antibiotic resistance) to differentiate transformed cells from those not transformed is called selection. Because bacteria reproduce asexually and are immobile on solid media, it is likely that the hundreds of thousands of bacteria making up that colony are genetically identical daughters of a single cell. This allows us to take bacteria from a single colony and sub-culture them in liquid media to make millions of identical copies. However, knowing that the bacteria growing in your broth or on your agar with ampicillin all have the plasmid responsible for amp resistance does not confirm that these bacteria also have our gene of interest insert. There are a small proportion of bacteria on your selection plates that may have a plasmid lacking the gene of interest. That can happen when a vector plasmid is "empty". Our plasmid, pPD129.36 ''lsy-2'', was genetically modified from a purchased base plasmid genetically synthesized by a pharmaceutical company. Remember that researchers need to be able to study any gene of interest; therefore, base plasmids are created that allow insertion of any gene of interest in proper alignment with synthetic gene promoters so that the gene of interest can be expressed as desired in a bacterial or a yeast model system. The first step in this process after acquiring a base plasmid (such as pPD129.36) is to isolate small segments of chromosomal DNA that contain your gene. Then you must make lots of copies of your gene by PCR after designing primers that will copy it. The next step is to ligate (insert) the gene from the PCR product into the vector plasmid so that it is in proper alignment with the promoters. If all goes well the ligation works and you achieve this proper alignment and after transformation (uptake of the plasmid by a cell). both the selection gene and the gene of interest are expressed. However, to do a ligation you must cut the vector plasmid at specific places in the DNA using specific restriction enzymes and then reanneal the ends after the gene of interest is inserted. Occasionally that ligation/annealing process fails to work properly and the plasmid DNA anneals back on itself. We need a way to find only the colonies with successful ligation that are expressing our ''C. elegans lys-2 gene'' off the synthetic vector plasmid.<br> | ||
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To | To confirm that the ''E coli'' bacterial colony you picked has lys-2 we are going to do a '''colony PCR'''. Instead of adding purified ''lsy-2'' gene fragments as template DNA in a PCR reaction with primers specific for your gene, you will add a TINY little part of a colony as the template for your PCR reaction. During the first heat cycle the cells will burst open and release their DNA into the reaction. We will test both well-isolated, non-satellite colonies per group to be sure we continue with bacteria that have the gene of interest inserted in our plasmid.<br> | ||
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#Obtain three PCR tubes and lids from your instructor in your team color. | #Obtain three PCR tubes and lids from your instructor in your team color. | ||
