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Changing Environment Long Term
General Procedures |
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< | Inoculating Plates |
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> | Inoculating Tubes |
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< | 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. |
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> | To start an experiment, obtain 98 test tubes. Split the test tubes into two racks, 49 in each. Label tubes 1-16, and fill them with 3 ml 0.01% glucose. Inoculate the tubes with 5 µl of frozen stock, starting with 1-1, with a red (Ara-) strain. In the next tube, 1-2, inoculate with a white (Ara+) strain. Continue this pattern in each row of both racks, leaving one tube blank. Grow for two full days at 37°C and 120 rpm. |
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< | 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. |
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> | In order to fill the test tubes with 3 ml of media, use the repeat pipettor with a 50 ml syringe. Set the pipettor to setting 3 to aliquot 3 ml each time. To use the 50 ml syringe, the gray plastic extending piece of the repeat pipettor must be attached to the syringe. |
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| | Preparing Media |
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< | 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. |
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> | Growth media should be made by adding the necessary nutrients to 500 ml bottles of DM0 and can be stored indefinitely. For 0.01% Glucose or Galactose, add 125 µl of 40% Glucose or Galactose to 500 ml DM0. Fill tubes according to the schedule. |
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< | 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. |
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> | Check bottles of media for contamination by swirling them before filling new tubes. Contamination is marked by cloudiness or discoloration of the media. Also be sure to use new troughs each time filling new tubes. |
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< | Transfering Plates |
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> | Transferring Tubes |
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< | 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. |
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> | Every 24 hours, transfer 11.7 µl from each tube into 3 ml of fresh growth medium using the P-20 pipette. This is a 256x dilution. Be sure to break the meniscus of the fresh media with the pipette tip before expelling the sample. This one day cycle of dilution and growth is one time increment in the experiment. |
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< | 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. |
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> | Every 16 transfers, freeze all 96 samples for later evaluation. |
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| META TOPICMOVED |
by="MarkKauth" date="1211229545" from="Lab.ProceduresEvolvabilityLongTerm" to="Lab.ProceduresChangingEnvironmentLongTerm" |
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