IGEM:IMPERIAL/2009/M3/Assays/tempshift2

=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.