---++ Population Genetics Long Term ---+++ Daily Procedure *Supplies* 1 13 x 50 ml flasks filled with 9.9 ml of DM500 (DM0 supplemented with 0.05% glucose). 1 12 x tetrazolium arabinose (TA) plates. 1 12 x wet DTs (test tubes filled with 9.9 ml of saline). 1 80% glycerol, sterilized. 1 12 x 15 ml conical orange-capped tubes. *Procedure* 1 Count TA plates from previous day. Plate counts should total 400-500 red+white colonies. 1 Transfer 100 µl from each of the previous day's flasks to a new DM500 flask. Vortex each for at least 5 seconds to mix. 1 Transfer 10 µl from each new flask to a wet DT to make a 10<sup>3</sup> dilution. 1 Place new flasks in a 37°C incubator shaking at 120 rpm. 1 Plate 50 µ from each wet DT on a TA plate. 1 Add 1.6 ml of 80% glycerol to each flask from the previous day. Use the repeat pipettor set to "4" with the 10 ml tip twice. Vortex each flask for at least 5 seconds to mix. 1 Pour the entire contents of each flask from the previous day into a 15 ml conical tube and freeze at -80°C. ---+++ Notes * To begin the experiment inoculate two sets of flasks with entire colonies grown on MG plates. Do not use TA plates as this causes higher cell numbers for Ara<sup>+</sup> strains. * Each frozen culture has enough DNA for two standard preps of 5 ml yielding 10-40 µg of genomic DNA each.
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Topic revision: r1 - 2008-05-27 - JeffreyBarrick