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MIT7_13f08_lab10_Protocol_Generating - MIT OpenCourseWare...

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7 . 1 3 F a l l 2 0 0 8 P a g e | 1 Generating and screening for mutants in Fur Concept Growth on manganese rich media has been shown in both E. coli and PAO1 to select for mutants with defects in iron acquisition. The majority of these mutants are in the fur gene. In Pseudomonas, Fur is essential for aerobic growth, so any mutants isolated are point mutations, rather than knockouts. Media LB 1. Add 25 g LB to 1 L deionized water. For plates, add 15 g bacto agar. 2. Autoclave for 35 min on the fluid cycle. 3. Let cool, then add required antibiotic for selective media. If making plates, pour into petri dishes, let solidify, and refrigerate. Manganese plates: 2 g Tryptone 1.2 g NaCl 10mM MnCl2 7.5 g agar 500 ml deionized water Autoclave and cool to 50 o C, then add 50 uM desferral (desferroxamine) (make and filter sterilize a 25 mM stock solution. Use 2 ul/ml) These plates must be made fresh – use within a week.
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