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 l 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 l from each new flask to a wet DT to make a 103 dilution.
  4. Place new flasks in a 37C incubator shaking at 120 rpm.
  5. Plate 50 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 -80C.

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 g of genomic DNA each.
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Topic revision: r1 - 2008-05-27 - 04:33:53 - Main.JeffreyBarrick
 
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