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IPTG test

IPTG is a molecular mimic for allolactose therefore it triggers the expression on Plac allowing the expression of the gene under the control of this promoter (RFP or T4 lysis cassette in our case).

• shake gently an overnight colture of bacteria to resuspend cells (you can do it inverting the tubes until the solution is homogeneous)
• take an aliquot of 100 microL and transfer to a new eppendorf tube
• add 900 microL of new medium (LB + antibiotic); this will be a dilution 1:10
• spot the cells (100 microL) in a multi-well: 2 wells per colony (the second is the negative control)
• for every colony: in one well add IPTG (500 microM) and in the second well add the same volume of water (negative control)
• cover each well with 20 microL of mineral oil (Chill-Out Liquid Wax)

Move to the plate-reader and remind to start it up 10' before starting

• open the software Gen5
• use the old file from IGEM but remember to save it as a new file (in order to be able to modify it without deleting the old settings)
• double click on procedure
• set temp at 37°C
• in "start kinetic" set:
  • time: 12 hours
  • interval: 10 minutes
  • read (for the : keep the settings like that (absorbance, endpoint, normal) and put 600 nm as wavelength