(r4) Barrick Lab :: ProceduresChangingEnvironmentLongTerm

---+++ 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.

---++++ Plate 1

Red Plate. Strains 606/607. 8 Replicates, one per column.
| 1 | 1 | D-Glucose |
| 2 | 2 | Glycerol |
| 3 | 3 | &beta;-Lactose |
| 4 | 4 | D-Fructose |
| 5 | 5 | D-Ribose |
| 6 | 6 | D-Galactose |
| 7 | 7 | L-Rhamnose |
| 8 | 8 | Melibiose |
| 9 | 9 | D-Gluconic Acid |
| 10 | 10 | D-Sorbitol |
| 11 | 11 | Acetate |
| 12 | 12 | L-Malic Acid |

---++++ Plate 2

White Plate. Strains 606/607. 8 Replicates, one per column.
| 1 | 13 | Pyruvic Acid |
| 2 | 14 | DL-Lactic Acid |
| 3 | 17 | Thymidine |
| 4 | 18 | Inosine |
| 5 | 19 | D-Trehalose|
| 6 | 20 | D-Serine |
| 7 | 21 | D-Glucuronic Acid |
| 8 | 22 | L-Alanine |
| 9 | 23 | D-Glucosamine |
| 10 | 37 | Glucuronamide |
| 11 | 71 | D-Arabinose |
| 12 | 72 | L-Proline |

---++++ Plate 3

Red Plate. Strains 606/607. 8 Replicates, one per column.
|  | Treatment | Days |
| 1 | L-Proline, Thymidine, Acetate, D-Glucose | 1 |
| 2 | L-Proline, Thymidine, Acetate, D-Glucose | 2 |
| 3 | L-Proline, Thymidine, Acetate, D-Glucose | 4 |
| 4 | L-Proline, Thymidine, Acetate, D-Glucose | 8 |
| 5 | L-Proline, Thymidine, Acetate, D-Glucose | 16 |
| 6 | L-Proline, Thymidine, Acetate, D-Glucose | 32 |
| 7 | Pyruvic Acid, D-Galactose, &beta;-Lactose, D-Glucose | 1 |
| 8 | Pyruvic Acid, D-Galactose, &beta;-Lactose, D-Glucose | 2 |
| 9 | Pyruvic Acid, D-Galactose, &beta;-Lactose, D-Glucose | 4 |
| 10 | Pyruvic Acid, D-Galactose, &beta;-Lactose, D-Glucose | 8 |
| 11 | Pyruvic Acid, D-Galactose, &beta;-Lactose, D-Glucose | 16 |
| 12 | Pyruvic Acid, D-Galactose, &beta;-Lactose, D-Glucose | 32 |

---++++ Plate 4

White Plate. Strains 606/607. 8 Replicates, one per column.
|  | Treatment | Days |
| 1 | L-Rhamnose, D-Galactose, D-Fructose, D-Glucose | 1 |
| 2 | L-Rhamnose, D-Galactose, D-Fructose, D-Glucose | 2 |
| 3 | L-Rhamnose, D-Galactose, D-Fructose, D-Glucose | 4 |
| 4 | L-Rhamnose, D-Galactose, D-Fructose, D-Glucose | 8 |
| 5 | L-Rhamnose, D-Galactose, D-Fructose, D-Glucose | 16 |
| 6 | L-Rhamnose, D-Galactose, D-Fructose, D-Glucose | 32 |
| 7 | Acetate, D-Ribose, Glycerol, D-Glucose | 1 |
| 8 | Acetate, D-Ribose, Glycerol, D-Glucose | 2 |
| 9 | Acetate, D-Ribose, Glycerol, D-Glucose | 4 |
| 10 | Acetate, D-Ribose, Glycerol, D-Glucose | 8 |
| 11 | Acetate, D-Ribose, Glycerol, D-Glucose | 16 |
| 12 | Acetate, D-Ribose, Glycerol, D-Glucose | 32 |

---++++ Plate 5

Red Plate. Strains 606/607. 8 Replicates, one per column.
|  | Treatment | Days |
| 1 | Random 1 | 1 |
| 2 | Random 2 | 1 |
| 3 | Random 3 | 1 |
| 4 | Random 4 | 1 |
| 5 | Random 5 | 1 |
| 6 | Random 6 | 1 |
| 7 | Random 7 | 1 |
| 8 | Random 8 | 1 |
| 9 | Random 9 | 1 |
| 10 | Random 10 | 1 |
| 11 | Random 11 | 1 |
| 12 | Random 12 | 1 |

-- Main.JeffreyBarrick - 24 Sep 2007
Barrick Lab > ProceduresChangingEnvironmentLongTerm
Contributors to this topic
JeffreyBarrick, MarkKauth
Topic revision: r4 - 2007-11-01 - 23:59:44 - Main.JeffreyBarrick