---++Protocol for Determining Mutation Rate to Phage (T5) resistance ---++++Day -2: Prepare Media/Revive/Titer Phage 1. If necessary, revive the frozen stock of each strain in 10 ml of LB in a 50 ml flask by scraping the top of the strain to be tested. 2. Grow the primary culture 16-24 hours at 37°C. 3. Prepare 27<sup>1</sup> MG plates per condition to be tested. ---++++Day -1 : Precondition * Transfer a 10,000 fold dilution<sup>2</sup> from the primary culture into a flask of 10 ml of DM25<sup>3</sup>(or the medium that will be used for the fluctuation test) that will serve as the secondary culture. Grow 16-24 hours at 37°C. ---++++Day 0: Growth of Independent Cultures * Transfer between 100-1000 cells into 24 replicate lines using a 96-well plate. Transfer ~1000 fold dilution into 3 separate flasks (to serve as the control). Grow between 16-24 hours at 37°C. ---++++Day 1: Plating 1. Plate the entirety of each separate replicate line in the 96-well plate onto an MG + phage plate (the amount of phage you will plate will differ depending on the titer; use an excess amount relative to the expected final density of the culture). 2. Plate a ~10<sup>6</sup> dilution (or a dilution to get a reasonable colony count) of the control lines on MG plates. 3. Grow ~48 hours. ---++++Day 3: Counting * After ~48 hours of growth, count the number of colonies on each plate. ---++++Notes <sup>1</sup>Each strain needs to be plated ~3 times on non-selective medium to determine final density. <sup>2</sup>In this case, we preconditioned with LB media, which grows to 100 times the density of DM25 media. To get 100 fold growth, we did a 10<sup>4</sup> dilution. <sup>3</sup>The appropriate growth medium should be used such that the cultures can grow to the correct final density in ~100-200 microliters of medium.
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Topic revision: r2 - 2009-01-20 - WaldoDittmar