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BISC219/F12: RNAi Lab 7

46 bytes added, 08:53, 10 October 2012
Picking a Bacterial Colony
== Picking a Bacterial Colony ==
Your instructor will give each pair an LB+amp ampicillin plate on which bacteria containing our pPD129.36 ''lsy-2'' plasmid are selectively growing. Please pick two well isolated colonies, circle them, label one A and the other B and write your initials and group color on the agar side of the plate. <br><br>
How can you be sure that your colonies contain the plasmid carrying the gene you are interested in studying? In theory, any colony of bacteria growing on your LB+amp plate should contain a plasmid because the gene for antibiotic resistance is not chromosomal, but instead expressed from your plasmid. Because only transformed bacteria are resistant to ampicillin, if we grow the bacteria on or in a medium containing ampicillin, those bacteria that did not take up plasmid DNA should not be able to reproduce to form colonies while those that express plasmid gene products and transfer the plasmid to their progeny will form colonies. The amp resistance gene on the plasmid encodes an enzyme called beta-lactamase. This enzyme is a secreted, soluble protein, which means that there may be smaller, non-transformed, "satellite" colonies around a true transformant. This happens because the ampicillin in the media is destroyed in the area immediately around the colony secreting the enzyme; therefore, there is no ampicillin in the area around the transformant and non-transformed cells can grow and divide enough to form smaller, satellite colonies. You must be careful to pick ONLY the bigger, central colony and not the satellites. The satellite bacteria are unlikely to carry the plasmid that contains the ''C. elegans lyslsy-2'' gene.<BR><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. Since all of that previous work to make ''pPD129.36 lyslsy-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 lyslsy-2 gene'' into the vector plasmid.<br>
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To confirm that the ''E coli'' bacterial colony you picked has ''lyslsy-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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# Obtain a previously prepared culture of ''E. coli '' bacteria that have been transformed with pPD129.36 lys''lsy-2 and '' growing on solid Luria broth medium with ampicillin. Select two large, well isolated colonies and label them A and B on the bottom of the plastic plate using your fine tipped Sharpie. Label the plate on the bottom with your initials, your lab day, and add a small strip of your team color tape.
#Obtain three PCR tubes and lids from your instructor in your team color.
#Label the '''side ''' of each pcr PCR tube A, B and C - the marker WILL rub off the top. Tube C will serve as your negative control with no colony added.
#Add 30 ul of master mix to each tube. Your master mix will include: 23 μL H<sub>2</sub>O; 3 μL of PCR buffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl2 pH 8.3); 0.67 μL of 10 mM dNTPs; 0.67 μL of forward primer (20 μM stock); 0.67 μL of reverse primer (20 μM stock); 2 units/μL Taq polymerase
#After each tube has master mix, use the sterile end of an autoclaved toothpick (''not'' the end you are touching) or the end of a sterile micropipet tip and gently touch the center of your colony of interest and pick up a tiny, barely visible amount of the bacteria. DO '''NOT TAKE THE ENTIRE COLONY!!!''' Make sure that you select colonies that are large enough to have a significant amount remaining bacteria after taking your sample.
#Repeat for colony B
#Snap the lid on the tubes, pulse them briefly in the microcentrifuge with the appropriate rotor and adapter and bring them to the thermal cycler for PCR initiation.
#Finally, seal the plate with parafilm. Invert it (agar side upso condensation will not drip on your colonies) and store it in the refrigerator in your lab day's rack until the day before the next lab when you will set up an overnight culture from a colony that is positive for ''lyslsy-2''.
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