By David M. Prescott (Ed.)
ISBN-10: 0125641087
ISBN-13: 9780125641081
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Additional resources for Methods in Cell Biology, Vol. 8
Example text
The mutant cells survive the treatment and are isolated. Auxotrophic mutants have been extremely useful in microbial genetic analysis. They offer possibilities for many kinds of experiments in which specifically selective media can be used to isolate desired kinds of clones. Many of these mutants have been analyzed biochemically and shown to involve a single gene mutation resulting in absence or malfunction of a specific enzyme (Wagner and Mitchell, 1964). This feature is of particular importance in mammalian cells since various drug-resistant animal cell DEFICIENT CELL \ MINIMAL MEDIUM + BUdR SUFFICIENT CELL @--- ENRICHED MEDIUM @ COLONY FIG.
Both the cylinder and the grease have been previously sterilized by autoclaving. When the cylinder is securely placed on the bottom of the plate, a few drops of trypsin are added to the cylinder. After a few minutes at room temperature, the cells begin to round up. Using asterile Pasteur pipette with an attached rubber bulb, the cells in the cylinder are released by gentle pipetting and are transferred to a new plate containing F12FClO medium. An experienced person can easily pick about 60 clones in an hour.
D. Clone Isolation, Testing, and Auxotrophic Analysis Day I6 Day 19 Day24 Pick the surviving colonies developed in each plate (see Section 111, B for clone isolation technique). With an effective mutagen, about 0-5 colonies should be found per plate. Incubation should continue until each colony contains 200 or more cells. Isolate 100-150 clones, and transfer each to a 35 mm plate containing complete growth medium. These clones are allowed to grow for 3 additional days to obtain enough cells for testing.
Methods in Cell Biology, Vol. 8 by David M. Prescott (Ed.)
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