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BISC219/F12: RNAi Lab 7

17 bytes added, 08:53, 23 October 2012
Picking a Bacterial Colony
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 L4440) 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. Since all of that previous work to make ''pPD129.36 lsy-2'' was done by previous researchers, you will begin our project by making sure that you are starting with a bacterial colony that successfully ligated ''C. elegans lsy-2 gene'' into the plasmid.<br>
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== Colony PCR ==
To confirm that the ''E coli'' bacterial colony you picked has ''lsy-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 (as it was done to make the plasmid), 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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