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Team:Evry/Protocols/06
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Revision as of 11:01, 26 August 2013
Tecan Analysis
Principle
Tecan is used to characterized bacteria strain: bacteria growth, protein production, etc. The software measure the Optical Density (OD) or the fluorescence of a compound of interest (e.g. a protein fused with GFP) regurlarly, thus allowing to obtain the cinetic of phenomenas. With a 96 (8x12) wells plate, different conditions could be test.
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) |
|---|---|---|
| CaCl2 (1M) | 5 µL | |
| MgSO4 (1M) | 100 µL | |
| Glycerol (50%) | 10 mL | |
| 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
