215-Lab6B-Bacteria_ONs - Lab 6B- Making ON Bacterial...

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Unformatted text preview: Lab 6B- Making ON Bacterial Cultures Lab 6B Setting up Overnight Cultures of Bacteria You will need to come into the lab on Monday Oct. 18, between 7:00 -9:00 AM or 11:00 AM to 1:00 PM to set up the ON cultures of your bacterial transformants. 1. Background: Setting up bacterial ON cultures: As described in Lecture, DNA fragments in a plasmid vector. A plasmid, in turn, is carried in a bacterial E. coli host strain under conditions that select for the plasmid, i.e., by growing cells containing a plasmid in a medium containing an appropriate antibiotic. In our case, the plasmid contains an ampicillin resistance gene. Therefore, to select for the presence of this plasmid we must grow bacteria harboring this plasmid in the antibiotic ampicillin. Without positive selection for the plasmid (growth in LB + Amp broth), the bacteria may lose the plasmid, resulting a low yield of plasmid DNA when you perform plasmid minipreps next week. When a fragment of interest is required for experimentation, the vector can be purified from the E. coli host. The first step in this process is to generate an ON culture of the bacterial strain containing the vector. When working with bacteria, the key to sterile technique is to avoid prolonged exposure to the air. Do not leave plates, media bottles, or their caps open to the air for longer than necessary. Although flaming the bottle tops and pipets is a common practice in some labs, flaming is not essential if you work carefully, cleanly and efficiently. Cover your pipet tip racks after you are through using them. Remember that once a pipet or pipet tip has been laid down, it should no longer be considered sterile. Always examine your media solutions before you use them. If your media is cloudy or you see a fungi puff growing in it, do not use it! Throw it away by washing it down the sink. If you have the least suspicion that a solution or pipet is not sterile, do not use it. All plates, tips, tubes, pipets, etc. must be disposed in biohazard bags to be autoclaved! We will supply you with sterile pipets, tubes, solutions, and plates. It is your responsibility to keep your stocks sterile. 6B-1 Lab 6B- Making ON Bacterial Cultures 2. Each person will prepare ON cultures of the bacteria transformed with their mutant plasmids. 1. Label 2 culture tubes with your mutant clone names (e.g. T2JS2.09, T2JS5.09) It is best to write directly on the side of the sterile glass culture tube using the thick blue marker in your bench drawer. Do not write on the caps! Caps can be switched, causing strains to be mixed up. 2. Into each of two sterile capped reusable glass culture tubes, add 2.5 ml of LB +100 µ g/ml ampicillin solution. a) Remove the cap from a sterile culture tube. Work quickly to minimize contact of the tube with the possibly contaminated air. b) Use a sterile 10 ml pipette to draw up 5 ml of LB-Amp from the 5 ml tube that was made in the previowu lab. c) Transfer 2.5 ml of LB-amp into each of the tubes. 3. Inoculate the media with a colony from your bacterial transformation plate of each of your mutants. Inoculate each tube with a single bacterial colony by touching a sterile wooden stick to the colony, making certain that some of the cells have been transferred to the stick, and then dipping the stick into the liquid and shaking it a bit. Replace the tube's cap as soon as inoculation is complete. 4. Grow overnight at 37ºC with aeration. E. coli grow most efficiently at 37ºC with strong aeration. Place each culture into the 37ºC shaking incubator and allow them to incubate overnight (≥12 hours). A freshly saturated culture will be at a density of ~109 cells/ml and will look visibly turbid. An overnight culture can be stored at 4°C for several weeks without significant loss of viability. The next morning, the lab instructors will take your saturated cultures out of the incubator and put them at 4ºC (in your refrigerator) until next week. 6B-2 ...
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