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).
2. 12 x tetrazolium arabinose (TA) plates.
3. 12 x wet DTs (test tubes filled with 9.9 ml of saline).
4. 80% glycerol, sterilized.
5. 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.
2. 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.
3. Transfer 10 µl from each new flask to a wet DT to make a 10³ dilution.
4. Place new flasks in a 37°C incubator shaking at 120 rpm.
5. Plate 50 µ from each wet DT on a TA plate.
6. 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.
7. 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⁺ strains.
• Each frozen culture has enough DNA for two standard preps of 5 ml yielding 10-40 µg of genomic DNA each.