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Team:University of Sheffield /Wet Lab
From 2008.igem.org
Protocols
Tecan Fluorescence Measurement
Protocol used for characterization :
1. Grow 5ml of overnight cultures of DH5-alpha cells containing the plasmid with GFP-LVA.
2. Resuspend the overnight cultures in 50 ml of LB medium until the OD600 reaches about 0.6.
3. Quickly add 0.2 mM of IPTG to the medium and plate that into 96 well plate.
4. To 96 well plate add 180 ul of LB + IPTG as a control, 180 ul of DH5-alpha cells not induced with IPTG, finally add 180 ul of DH5-alpha induced with IPTG. Carry out the aforementioned in duplicates.
5. Use the high flow cytometry machine Tecan to measure the fluorescence every 15 minutes for 8 hours (excitation wv – 485 nm, emission wv- 535 nm )(including shaking = aeration of the cultures 2 minutes prior each measurement)
