General Procedures

Inoculating Plates

To start an experiment, thaw a plate that has a checkerboard pattern of Ara+ and Ara- cells established from the freezer. Save the plate used to inoculate the culture, as it will serve as the time zero reference for the entire evolution experiment.

All long term evolution plates should have a checkerboard pattern of Ara+ and Ara- marked cells. Red plates have Ara- in well A1, and white plates have Ara+ in well A1. This is so that contamination between wells can be detected.

Preparing Media

Fill several deep 96-well microplates for each transfer at the same time with 900 µl of growth medium using the Biolog multichannel repeat pipettor. Growth media should be made by adding the necessary nutrients to 500 ml bottles of DM0 and can be stored indefinitely.

The largest contamination risks seem to be in the stock solutions that are used to fill the plates and the troughs used to fill the multichannel pipettor. Check bottles of media for contamination by swirling them before filling new microplates. Examine troughs for any sign of white, yellow, or black cloudiness. Better yet -- use new troughs each time. Finally, before using a new batch, check one microplate for smaller amounts of contamination by using the pin tool to deposit 3.5 µl from each well on a large square TA plate.

Transfering Plates

Every 48 hours, transfer 3.5 µl from each well into 900 µl of fresh growth medium using the deep 96-well pin tool. This two day cycle of dilution and growth is one time increment in the experiment.

Every 4 transfers, take the new microplate (that you have just transfered to) and use the pin tool to deposit 3.5 µl drops of the culture on a large square TA plate to check for contamination.