iGEM Headquarters is conducting a study to explore the question, "How close can the numbers be when fluorescence is measured all around the world?" Teams were given three test devices of green fluorescent protein (GFP) under different constitutive promoters, as well as a positive and a negative control. We were asked to measure the fluorescent output of these samples using equipment in our lab.
The MIT team measured these samples on our flow cytometer and reported the results to iGEM HQ for further analysis and comparison to other teams' results.
E.Coli Flow Cytometry Protocol
via Nicholas DeLateur at the Weiss Lab
A single isogenic colony is picked up with a sterile pipette tip and used to inoculate a well in a 96-well plate containing 200 uL of M9-Glyc-Kan media. The 96-well plate is shaken at 900 RPM at 37 C for 12-16 hours overnight.
1. Each well is diluted twice, 15 uL into 185 uL of M9-Glyc-Kan.
2. The subculture is grown at 900 RPM at 37 C for 3 hours.
3. The subculture is diluted 5 uL into 145 uL of M9-Glyc-Kan and grown for 5 hours at 900 RPM, 37 C.
4. Transfer 5 uL of the subculture to 195 uL of 1 X PBS with 2 mg/mL Kanamycin.
5. Allow to sit on bench for 1 hour.
6. Run flow Cytometry.