Team:Evry/Protocols/06

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<h2>TECAN</h2>
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<h3>Medium preparation</h3>
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<p>
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For the Tecan analysis we must use M9 medium and not a classical LB medium because turbidity and fluorescence of our sample are measured. Then, in order to obtain good results, the medium must not emit a side signal, that is why we use the M9 medium.
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</p>
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<p>
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<u>Composition for 50 mL of:</u>
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</p>
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<table cellpadding="10" cellspacing="0" align='center' border="1">
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<thead>
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  <tr>
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  <th align="center">
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    Reagent
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  </th>
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  <th align="center">
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    M9 medium (without iron)
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  </th>
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  <th align="center">
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    M9 medium (with iron)
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  </th>
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  </tr>
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</thead>
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<tbody>
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  <tr>
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  <td align='center'>
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    CaCl<sub>2</sub> (1M)
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  </td>
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  <td colspan="2" align='center'>
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    5 µL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    MgSO<sub>4</sub> (1M)
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  </td>
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  <td colspan="2" align='center'>
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    100 µL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    Glycerol (50%)
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  </td>
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  <td colspan="2" align='center'>
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    10 mL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    Thiamine
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  </td>
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  <td colspan="2" align='center'>
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    5 µL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    NaOH (pH 7.4)
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  </td>
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  <td colspan="2" align='center'>
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    12.5 µL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    H<sub>2</sub>O
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  </td>
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  <td align='center'>
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    40 mL
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  </td>
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  <td align='center'>
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    39 mL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    FeSO<sub>4</sub> (10mM)
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  </td>
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  <td align='center'>
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    -
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  </td>
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  <td align='center'>
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    50 µL
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  </td>
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  </tr>
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  <tr>
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  <td align='center'>
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    Casamino acids (0.2%)
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  </td>
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  <td align='center'>
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    -
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  </td>
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  <td align='center'>
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    1 mL
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  </td>
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  </tr>
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</tbody>
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</table>
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<p>
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Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.
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</p>
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<h3>TECAN analysis</h3>
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<p>
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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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</p>
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<p>
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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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</p>
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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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</ul>
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<p>
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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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</p>
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<p>
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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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</p>
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<p>
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After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).
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</p>
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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>
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 +

Revision as of 08:40, 26 August 2013

Iron coli project

TECAN

Medium preparation

For the Tecan analysis we must use M9 medium and not a classical LB medium because turbidity and fluorescence of our sample are measured. Then, in order to obtain good results, the medium must not emit a side signal, that is why we use the M9 medium.

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.

TECAN analysis

Using the TECAN analysis, we are measuring the capacity of repression of the natural binding site that we extract from the E.coli's genome.

Preculture with M9 medium (with carbenicillin) have been launched for BL21 transformed with our 1st construction:

  • Natural Fur Binding Site of Fec A + sfGFP (clone 1, 2, 3)
  • Natural Fur Binding Site of Fep A + sfGFP (clone 1, 2, 3)
  • Natural Fur Binding Site of Ace B + sfGFP (clone 1, 2, 3)

Note: Preculture have been made in M9 medium with iron in oder to inhibit the expression of sfGFP.

After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).

After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).

AJOUTER DETAIL DES CYCLES

Tecan Analysis

Principle

Preparation

Analysis

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