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| + | <h4 class=title4> <FONT COLOR="blue">Preparation of antibiotic stocks </FONT></h4> |
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| - | <h4 class=title4> <u> Preparation of antibiotic stocks </u> </h4> | + | |
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| - | <table> | + | <table border="1"> |
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| - | <td> <b>Antibiotic </b> </td> | + | <td> <b><FONT COLOR=#000033>Antibiotic</font> </b> </td> |
| - | <td> <b>Stock concentration </b> </td> | + | <td> <b><FONT COLOR=#000033>Stock concentration</font> </b> </td> |
| - | <td class= table> <b>Protocol </b> </td> | + | <td class= table> <b><FONT COLOR=#000033>Protocol </font></b> </td> |
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| | <td> Amplicilin </td> | | <td> Amplicilin </td> |
| - | <td class= conc> 50 mg/mL </td> | + | <td class= conc> 100 mg/mL </td> |
| - | <td class= proto> Weight 1g of Amplicilin powder, solubilization into 40mL of miliQ water. <br>Vortex 5min and filtration with a 200nm filter. </br> Stock : -20°C (aliquots of 1mL) <br> </br> </td> | + | <td class= proto> Weight 1g of Amplicilin powder, solubilization into 20mL of miliQ water. <br>Vortex 5min and filtration with a 200nm filter. </br> Stock : -20°C (aliquots of 1mL) <br> </br> </td> |
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| - | <h6>08-16-2014</h6>
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| - | <h4> <u>Tests of antibiotics' stocks</u> </h4>
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| - | Six plates of LB agar were made.
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| - | Five of them contained one of those antibiotics in the dilution 1:1000:
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| - | <ul>
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| - | <li> Chloramphénicol
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| - | <li> Kanamycin
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| - | <li> Erythromycin
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| - | <li> Ampicilin
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| - | <li> Tetracyclin
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| - | </ul>
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| - | (The last one was the control of the growth of our bacteria without antibiotics)
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| - | We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.
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| - | <h4> <u>Survivability tests</u> </h4>
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| - | Two plates of MB 1X and M9 1X were made and divised in two parts.
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| - | Each plate was sowed with 5µL of a liquid MB 1X culture of Pseudovibrio denitrificans(dated 12th August) in a part and with 5µL of a liquid M9 1X culture Pseudovibrio denitrificans (dated 12th August) in the other part.
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| - |
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| - | <h4> <u>Pre-cultures</u> </h4>
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| - | Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C.
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| - | New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C.
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| - | <u>From glycerol stocks:</u>
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| - | <ul>
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| - | <li>Bl21
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| - | <li>Top10
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| - | <li>DH5a
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| - | </ul>
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| - | <u>From plates:</u>
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| - | <ul>
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| - | <li>DH5a tranformed with pCB1C3
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| - | <li>Top10 transformed with pQexp
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| - | <li>DH5a pyr tranformed with pMK2
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| - | </ul>
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| - | Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection.
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| - | For each medium we make a negative contrôle without bacteria.
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| - |
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| - | <h6>08-17-2014</h6>
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| - | <h4> <u>Tests of antibiotics' stocks - Results</u> </h4>
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| - |
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| - | <table border="1">
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| - | <tr >
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| - | <td> <b>Plates </b> </td>
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| - | <td>LB Agar only </td>
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| - | <td>LB+Cam </td>
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| - | <td>LB+Kan </td>
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| - | <td>LB+Amp </td>
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| - | <td>LB+Erm </td>
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| - | <td>LB+Tet </td>
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| - | </tr>
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| - |
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| - |
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| - | <tr>
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| - | <td> <b>E.coli</b> </td>
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| - | <td>X </td>
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| - | <td>0 </td>
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| - | <td>X </td>
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| - | <td>0 </td>
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| - | <td>X </td>
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| - | <td>0 </td>
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| - | </tr>
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| - | </table>
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| - |
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| - | There was a problem with the stock of Kanamycine so we made a new one at 25mg/mL.
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| - | For the erythrompycine, we learn that E. Coli is not really sensitive to this antibiotic and that it's better to use it with a dilution at 1:100
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| - |
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| - | <h4> <u>Survivability tests - Results</u> </h4>
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| - |
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| - | <table border="1">
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| - | <tr >
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| - | <td> <b>liquid cultures/Plates </b> </td>
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| - | <td> <b>MB </b> </td>
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| - | <td> <b>M9 </b> </td>
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| - | </tr>
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| - |
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| - | <tr >
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| - | <td> <b>MB </b> </td>
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| - | <td> <b>++++ </b> </td>
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| - | <td> <b>++ </b> </td>
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| - | </tr>
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| - |
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| - | <tr >
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| - | <td> <b>M9 </b> </td>
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| - | <td> <b>++ </b> </td>
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| - | <td> <b>+ </b> </td>
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| - | </tr>
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| - | </table>
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| - |
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| - | Any liquid culture can be plated and will grow up. Of course, it works better on MB medium, wich is a rich medium than on M9.
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| - |
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| - | <h6>08-18-2014</h6>
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| - | <h4> <u>Tests of antibiotics' stocks - Results(2)</u> </h4>
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| - | <table border="1">
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| - | <tr >
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| - | <td> <b>Plates </b> </td>
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| - | <td>LB Agar only </td>
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| - | <td>LB+Kan </td>
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| - | <td>LB+Erm (1:100)</td>
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| - | </tr>
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| - |
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| - |
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| - | <tr>
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| - | <td> <b>E.coli</b> </td>
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| - | <td>X </td>
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| - | <td>0 </td>
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| - | <td>0 </td>
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| - | </tr>
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| - | </table>
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| - |
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| - | New stocks are ready for antibiotics' tests on Pseudovribrio
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| - | <h4> <u>Plates for antibiotics's tests</u> </h4>
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| - | For each tested antibiotic we made the following range of concentrations:
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| - |
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| - | <table border="1">
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| - | <tr >
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| - | <td> <b>0 </b> </td>
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| - | <td> <b>1:500 </b> </td>
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| - | <td> <b>1:1000 </b> </td>
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| - | <td> <b>1:50 000 </b> </td>
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| - | <td> <b>1:100 000 </b> </td>
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| - | </tr>
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| - | </table>
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| - | We made these range two time to have a replicate and we added a controle plate without antibiotic on which we showed all our tested bacteria.
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| - | We made these same ranges of tested antibiotic's concentrations for three media:
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| - | <ul>
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| - | <li>MB agar 1X
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| - | <li>MB agar 0.5X
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| - | <li>M9 agar 1X
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| - | <ul>
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| - |
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| - | Each plate is made like in the following table:
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| - | <table border="1">
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| - | <tr >
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| - | <td> <b>/ </b> </td>
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| - | <td> <b>0 </b> </td>
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| - | <td> <b>1:500 </b> </td>
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| - | <td> <b>1:1000 </b> </td>
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| - | <td> <b>1:50 000 </b> </td>
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| - | <td> <b>1:100 000 </b> </td>
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| - | </tr>
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| - |
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| - | <tr >
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| - | <td> <b>Medium+agar </b> </td>
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| - | <td>20mL </td>
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| - | <td>20mL </td>
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| - | <td>20mL </td>
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| - | <td>20mL </td>
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| - | <td>20mL </td>
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| - | </tr>
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| - |
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| - | <tr >
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| - | <td> <b>Antibiotic </b> </td>
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| - | <td>0 </td>
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| - | <td>40µL of stock</td>
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| - | <td>20µL of stock</td>
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| - | <td>40µL of stock diluted 1/100</td>
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| - | <td>20µL of stock diluted 1/100</td>
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| - | </tr>
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| - |
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| - | </table>
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| - | And plates are organised like shown in the following picture:
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| - | <img src="https://static.igem.org/mediawiki/2014/7/71/EVRYSch%C3%A9maplates.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - | </p>
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| - | <span class="cd-date"></span>
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| - | </div>
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