Team:Evry/Protocols/06

From 2013.igem.org

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https://static.igem.org/mediawiki/2013/f/f8/Tecan.png' width="500px"/></div>
https://static.igem.org/mediawiki/2013/f/f8/Tecan.png' width="500px"/></div>
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Technical triplicate are made to see the variation induced by the manipulator.
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Technical triplicate are made to see the variation induced by the manipulator.<br/>
Biological duplicate are made to determine the natural variation of the process observed.
Biological duplicate are made to determine the natural variation of the process observed.
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<h2> Analysis </h2>
 
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<h3>TECAN analysis</h3>
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<h3>Cycles</h3>
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<h1>AJOUTER DETAIL DES CYCLES</h1>
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Using the TECAN analysis, we are measuring the capacity of repression of the natural binding site that we extract from the <i>E.coli</i>'s genome.
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<h2> Analysis </h2>
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Preculture with M9 medium (with carbenicillin) have been launched for BL21 transformed with our 1<sup>st</sup> construction:
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<ul>
 
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<li>Natural Fur Binding Site of Fec A + sfGFP (clone 1, 2, 3)
 
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<li>Natural Fur Binding Site of Fep A + sfGFP (clone 1, 2, 3)
 
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<li>Natural Fur Binding Site of Ace B + sfGFP (clone 1, 2, 3)
 
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<b>Note:</b> Preculture have been made in M9 medium with iron in oder to inhibit the expression of sfGFP.
 
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After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).
 
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After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).
 
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<div align="center"><img src='https://static.igem.org/mediawiki/2013/1/1a/Plan_Tecan_12.08.2013.png' width="500px"/></div>
 
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<h1>AJOUTER DETAIL DES CYCLES</h1>
 

Revision as of 09:42, 26 August 2013

Iron coli project

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

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