Flowcytometry

Flowcytometry protocol
M9 minimal media

In this project, we wanted to measure promotor activities and their leakage, but also testing the constructs
whether they work or not. Analysis of these constructs was done with the help of the FACS for measuring
fluorescence.

Flowcytometry protocol

Inoculate 10ml M9 minimal medium + antibiotics + 1ml of 2% casamino acids and incubate overnight at 37 °C.
The next day, the pre-heated erlenmeyers containing the 10ml M9 minimal medium+ antibiotics + 1ml of 2% casamino
acids of are inoculated until an OD600 of 0.01.
The FACS can be prepared for measurements with the protocol provided by Maarten Mols:
File:Flowcytometryprotocol.pdf
Flow cytometer used for measurements: BD FACSCanto™, REF: 337175
The graphs were made with the programme Cyflogic.

100μl suspension of the logfase cells will be used for measurements with the FACS.
Later on, when the celldensity increases too much, the cells will be diluted with M9 media.

The PBADaraC promotor needs to be induced by arabinose.
Preparation for 10% arabinose stock:
Add 1g of arabinose to 9ml milliQ and filter sterilize the stock.
Concentrations for induction can be varied from 0 to 1%.
See for more information: http://openwetware.org/wiki/Arabinose

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M9 minimal media

Component Concentrations
1x M9 salts
2mM MgSO4
0.1mM CaCl2
0.4% carbon source (e.g. glycerol, glucose, etc.)
In sterile H2O
Protocol
For 1L of media

500ml 2xM9 salts ∗
20ml 0.1M MgSO4 ∗
200μl 0.5M CaCl2 ∗
469.8ml sterile deionized H2O ∗ (if carbon source to be added)
479.8ml sterile deionized H2O ∗ (if no carbon source to be added, useful as a control for sugar metabolism)
Combine above solutions using sterile technique. (May notice some precipitation during preparation but precipitate
should go back into solution once volume is brought up to 1l with sterile H2O.)
Add antibiotic as appropriate and store at 4°C

∗ Can be obtained from the media room
http://openwetware.org/wiki/Endy:M9_medium/minimal

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