IGEM:IMPERIAL/2009/M3/Assays/tempshift2

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Effect of Temperature on killing activity

Assay

This is to check how the restriction enzyme activity changes when the temperature is shifted up. We plan to get a model of how activity varies with temperature.

Protocol

1) The bacterial cultures were grown overnight in LB (Luria-broth) (Miller, 1972). Supplements were added at the following concentrations where appropriate: tetracycline, 10 μg ml−1; ampicillin, 100 μg ml−1; 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (XGal), 0.04 mg ml−1.
2) Growth of cultures was monitored by measuring the optical density at 600 nm (OD600).
3) Cultures were grown until mid-log phase at 28 °C (from OD values)
4) Transferred to 28 °C, 30, 36, 39, 42 (various temperatures- enough to know activity variance with temperature), respectively, with both a control and a transformed culture
5) CFU counts(refer to Assays) taken of 0 min after temperature upshift and 180 mins after temperature upshift

An example of the results that would be obtained.
Killing table.jpg