MICROBIAL GENETICS DAYS FOUR TO SIX
DAY FOUR
On Day 4, you will use the P1 transducing lysates that you made on Day 3 to infect the recipient strain BW140. You will once again be using sterile technique to transfer cultures between tubes, and will plate bacterial cultures on Petri plates.
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You can save yourself some time in lab today by labeling all your Eppendorf tubes and large culture tubes (shown) during incubations.
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Today you need to separate cells from the media in which they have been grown. You will do this by spinning the Eppendorf tubes in the microcentrifuge. To adjust the speed to 6000 rpm, use the buttons marked with the arrows on the right side of the keypad.
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The Eppendorf tube containing the BW140 cells will look something like this before you centrifuge it.
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After centrifugation, the cells will "pellet" at the bottom of the tube, leaving the media (the "supernatant") above the cells. You will remove this supernatant before resuspending the cells in MC media.
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Your 30 minute incubation of your cultures will take place in the 37C warm room on the roller drum.
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While waiting for cultures, you will spot the P1 lysates on an LB Kan plate. What do you expect to see growing on this plate? Why?
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DAY FIVE
On Day 5, you will perform a second phenotypic characterization using indicator plates, though today you will be patching and characterizing your P1 transductants.
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Here are some P1 transductants obtained by the staff during our pre-semester training week. The P1 transducing lysate was made from strain "NM7" and used to infect BW140 cells as you did on Day 4.
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DAY SIX
On Day 6, you will analyze the results of your phenotypic characterization (similar to GEN Day 3) and will, with luck, purify an Ara-LacZ+ strain for use in the Recombinant DNA Methods module.
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