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MICROBIAL GENETICS DAY ONE, PART TWO
Today in lab, you will be performing transposon mutagenesis on an Ara+ strain of E. coli, selecting for KanR and screening for Ara- colonies, and streaking four control strains on indicator plates for use later in the module.
TRANSPOSON MUTAGENESIS
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You will incubate your phage and cells in glass test tubes on your bench top. "Small and "large" culture tubes are pictured here.
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When the protocol states "shake at 37C for 1 hr," you will place your cultures on the roller drum in the warm room. Always balance your tubes, and turn the roller drum back on after placing your culture tubes on it.
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SELECTING FOR KAN-R AND SCREENING FOR ARA- COLONIES
The pictures below depict how to spread a sample of a liquid bacterial culture onto a Mac Ara Kan plate. (You also practiced this skill during the 7.02 training laboratory!) Remember--this needs to be done using sterile technique to avoid contamination!
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On your bench, you will find 20 Mac Ara Kan plates, which have red agar.
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You will also find one LB Xgal Kan plate, which has yellow agar.
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Label each plate appropriately (with your bench letter and initials).
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To prepare for plating, turn on the gas supply at your bench, and light the Bunsen burner with your striker. Be aware of the flame, and keep your body and papers away!
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Place your plate near your burner, and have an ethanol jar (with lid) and glass spreader within reach. Ethanol can be obtained from containers in the fume hoods.
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Dip your spreader into the ethanol jar so that the spreader is coated with a thin layer of ethanol. (Note: we will use 95% ethanol for spreading, though this picture depicts 70% ethanol).
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Remove the lid from the Petri plate, placing it face down on the bench. Squirt the liquid to be plated onto the center of the agar.
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Pass the spreader through the flame quickly. This will ignite the ethanol on the spreader, sterilizing it. Allow the flame to go out by itself.
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To avoid killing the bacteria on the plate, cool your spreader by touching it to the arar in a part of the plate away from where you squirted the bacterial culture.
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Spread the liquid evenly over the surface of the plate. An easy way to do this is to move the spreader back and forth while turning the Petri plate with your other hand, as seen in these pictures. Be careful to hold the plate by the edges so you do not accidentally contaminate the agar with your fingers!
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After spreading the liquid, replace the lid on the Petri plate. Allow the plate to sit on your bench undisturbed until it is completely dry.
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Return the spreader in the ethanol jar to kill any bacteria that remain on its surface. Reflame the spreader before plating your next sample.
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After plating all your plates, turn them upside down, place them in a plastic bag, and incubate them in the 37C warm room overnight.
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STREAKING BACTERIA ON AGAR PLATES
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On your bench, you will find a Mac Ara Kan plate (purple and black stripe) and Mac Lac Kan (blue and black stripe).
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You will also find an LB Kan plate (black stripe) and an LB Ara Xgal Kan plate (black, purple, and orange stripes).
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Divide each plate into four sectors, and be sure to write your bench letter and initials on each plate.
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Use a sterile toothpick (shown here) to pick a colony from your TAs plate.
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Use sterile sticks (shown here) to streak out each inoculation. Use the diagram in your laboratory manual as a guide.
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Used sticks and picks can be placed in the appropriate disposal cans at your bench.
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