Difference between revisions of "Team:Pasteur Paris/Week 10"
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1 ml of 10% magnesium sulfate MgSO4 (autoclaved too) (1.5 g in 15 ml of H2O).<br /> | 1 ml of 10% magnesium sulfate MgSO4 (autoclaved too) (1.5 g in 15 ml of H2O).<br /> | ||
1 ml of 0.2% Thiamin (vitamin B1).</p> | 1 ml of 0.2% Thiamin (vitamin B1).</p> | ||
− | <p style="text-align:left">At the end the volume of Agar was exceeded (350 ml and not 333 ml). So we had to redo this | + | <p style="text-align:left">At the end the volume of Agar was exceeded (350 ml and not 333 ml). So we had to redo this experiment the next day.</p> |
<p style="text-align:center"><u>05-08-15 – Experiment: Preparation of M9 medium – VB, PV</u><br /> | <p style="text-align:center"><u>05-08-15 – Experiment: Preparation of M9 medium – VB, PV</u><br /> | ||
<u>Redaction by VB</u></p> | <u>Redaction by VB</u></p> | ||
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<p align="center"><u>06-08-2015 </u><u>–</u><u> </u><u>Experiment: Minimal and M9 medium for TPA Toxicity and BAP</u><u> </u><u>–</u><u> </u><u>VB, PV</u><u> </u><br /> | <p align="center"><u>06-08-2015 </u><u>–</u><u> </u><u>Experiment: Minimal and M9 medium for TPA Toxicity and BAP</u><u> </u><u>–</u><u> </u><u>VB, PV</u><u> </u><br /> | ||
<u>Redaction by VB</u><u> </u></p> | <u>Redaction by VB</u><u> </u></p> | ||
− | <p style="text-align:left"><strong><u>Aim:</u></strong> Have prepared M9 | + | <p style="text-align:left"><strong><u>Aim:</u></strong> Have prepared M9 medium and minimal medium for Petri dishes</p> |
<p style="text-align:left">We restarted the last two protocols but this time we included the agar for the future Petri dishes.<br /> | <p style="text-align:left">We restarted the last two protocols but this time we included the agar for the future Petri dishes.<br /> | ||
For the future experiments every medium will have the same concentration of sugar for the bacteria growth, and the test of the TPA toxicity. We began by a theoretical concentration for our Petri dishes: to see the saturation concentration of TPA we decided to do dilutions at 1/10 and 1/20. <br /> | For the future experiments every medium will have the same concentration of sugar for the bacteria growth, and the test of the TPA toxicity. We began by a theoretical concentration for our Petri dishes: to see the saturation concentration of TPA we decided to do dilutions at 1/10 and 1/20. <br /> | ||
− | This time we decided to make 1.5 l of | + | This time we decided to make 1.5 l of minimal medium<br /> |
<strong><u>Protocol:</u></strong><strong><u> </u></strong></p> | <strong><u>Protocol:</u></strong><strong><u> </u></strong></p> | ||
<p style="text-align:left"><strong><u>Solution 1: (salts)</u></strong><strong><u> </u></strong><br /> | <p style="text-align:left"><strong><u>Solution 1: (salts)</u></strong><strong><u> </u></strong><br /> | ||
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<p style="text-align:left"><strong><u>Solution 3: </u></strong><strong><u> </u></strong><br /> | <p style="text-align:left"><strong><u>Solution 3: </u></strong><strong><u> </u></strong><br /> | ||
500 ml of H2O </p> | 500 ml of H2O </p> | ||
− | <p style="text-align:left">We underestimated the time for agar to solidify, so we had solid agar in | + | <p style="text-align:left">We underestimated the time for agar to solidify, so we had solid agar in the <strong><u>test-tube</u></strong>. Moreover one of the flask was not sterile so we contaminated our medium. We decided to return at the first protocol (for 1L). </p> |
<p style="text-align:left">At the end of this journey the two media were ready but they did not have the last ingredients.</p> | <p style="text-align:left">At the end of this journey the two media were ready but they did not have the last ingredients.</p> | ||
<p style="text-align:left"> </p> | <p style="text-align:left"> </p> | ||
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<ol> | <ol> | ||
<li style="text-align:left">XbaI ans PstI enzymatic digest of BBa_K808030 and pDG011</li> | <li style="text-align:left">XbaI ans PstI enzymatic digest of BBa_K808030 and pDG011</li> | ||
− | <li style="text-align:left">Gel | + | <li style="text-align:left">Gel purification of the digested fragments on 0.8% agarose gel. </li> |
<li style="text-align:left">Ligation of pDG011 and BBa_K808030 using T4 DNA ligase. </li> | <li style="text-align:left">Ligation of pDG011 and BBa_K808030 using T4 DNA ligase. </li> | ||
− | <li style="text-align:left">Gel | + | <li style="text-align:left">Gel migration on 0.8% agarose gel. </li> |
<strong><img src="https://static.igem.org/mediawiki/2015/9/91/IGEM_Pasteur-gel--week-10.png" alt="k" width="292" height="455" /></strong><u> </u><br /> | <strong><img src="https://static.igem.org/mediawiki/2015/9/91/IGEM_Pasteur-gel--week-10.png" alt="k" width="292" height="455" /></strong><u> </u><br /> | ||
− | <strong><u>Figure 8:</u></strong> Gel | + | <strong><u>Figure 8:</u></strong> Gel for verification of ligation. <u> </u><br /> |
There is no band for the ligation sample. | There is no band for the ligation sample. | ||
</p> | </p> | ||
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<td width="74" valign="top"><p>808010</p></td> | <td width="74" valign="top"><p>808010</p></td> | ||
<td width="106" valign="top"><p>30</p></td> | <td width="106" valign="top"><p>30</p></td> | ||
− | <td width="88" valign="top"><p>9 | + | <td width="88" valign="top"><p>9.33</p></td> |
− | <td width="88" valign="top"><p>0 | + | <td width="88" valign="top"><p>0.75</p></td> |
<td width="71" valign="top"><p>0,3</p></td> | <td width="71" valign="top"><p>0,3</p></td> | ||
− | <td width="106" valign="top"><p>1 | + | <td width="106" valign="top"><p>1.87</p></td> |
− | <td width="109" valign="top"><p>0 | + | <td width="109" valign="top"><p>0.06</p></td> |
</tr> | </tr> | ||
<tr> | <tr> |
Revision as of 21:44, 17 September 2015
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Week 10 Toxicity and solubility of TPA (Terephthalic acid)
Preparation of M9 and minimal media Solution 2: (agar) Solution 3: (sugar)
When the three parts have been autoclaved, 2 more ingredients must be added to the medium aseptically: At the end the volume of Agar was exceeded (350 ml and not 333 ml). So we had to redo this experiment the next day. 05-08-15 – Experiment: Preparation of M9 medium – VB, PV Aim: Get culture medium for BAP1 Pfeifer cells. Protocol: (for 1 l medium) Agar was directly put in a bottle and salts were dissolved in a beaker with water. 6 g of Sodium Phosphate Na2HPO4.H2O When this flask was autoclaved some ingredients were added in aseptical conditions: 1 ml of 1M Magnesium sulphate MgSO4 (6.02 g in 50 ml H2O) A mistake was done yesterday but this time an other did too: The last ingredients were added but they were not sterile so the medium failed again. 06-08-2015 – Experiment: Minimal and M9 medium for TPA Toxicity and BAP – VB, PV Aim: Have prepared M9 medium and minimal medium for Petri dishes We restarted the last two protocols but this time we included the agar for the future Petri dishes. Solution 1: (salts) Solution 2: (agar) Solution 3: We underestimated the time for agar to solidify, so we had solid agar in the test-tube. Moreover one of the flask was not sterile so we contaminated our medium. We decided to return at the first protocol (for 1L). At the end of this journey the two media were ready but they did not have the last ingredients.
Enzymatic Assay of NB-EsteraseFigure 8: Gel for verification of ligation. There is no band for the ligation sample. Gibson Assembly
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