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Team:Evry/Protocols/06
From 2013.igem.org
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<h2> Goal </h2> | <h2> Goal </h2> | ||
| - | <p>Tecan | + | <p>Tecan is a plate reader that is able to characterize our bacteria of interest by measuring, for example, growth rate (OD) and/or protein production (GFP). The software saves the measurements over time, thus allowing to study the kinectics of the studied phenomena. With a 96 wells plate (8x12), we can test various conditions.</p> |
<h2> Preparation </h2> | <h2> Preparation </h2> | ||
Revision as of 15:35, 2 September 2013
Tecan Analysis
Goal
Tecan is a plate reader that is able to characterize our bacteria of interest by measuring, for example, growth rate (OD) and/or protein production (GFP). The software saves the measurements over time, thus allowing to study the kinectics of the studied phenomena. With a 96 wells plate (8x12), we can test various conditions.
Preparation
Medium preparation
LB medium emit a side signal. As turbidity (OD) and fluorescence of our sample are measured, M9 medium is use instead.
Composition for 50 mL of:
| Reagent | M9 medium (without iron) | M9 medium (with iron) |
|---|---|---|
| M9 salt (5X) | 10 mL | |
| CaCl2 (1M) | 5 µL | |
| MgSO4 (1M) | 100 µL | |
| Glycerol (50%) | 800 µL | |
| Thiamine | 5 µL | |
| NaOH (pH 7.4) | 12.5 µL | |
| H2O | 40 mL | 39 mL |
| FeSO4 (10mM) | - | 50 µL |
| Casamino acids (0.2%) | - | 1 mL |
Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.
Pre-culture preparation
In a 15 mL tube, add 2 mL of M9 medium and inoculate BL21 cells (expression strain) from glycerol
To inhibit the expression of sfGPF, use M9 with iron, instead of classical M9.
After one night of culture, refresh the precultures by diluting them 200 times in M9 medium (with iron and carbenicillin).
After 8 hours of culture, prepare your wells plate according to the following scheme:
Technical triplicate are made to see the variation induced by the manipulator.
Biological duplicate are made to determine the natural variation of the process observed.
Cycles
AJOUTER DETAIL DES CYCLES
Analysis
Exemple à modifier
