Team:Lyon-INSA/notebook

From 2012.igem.org

(Difference between revisions)
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                       </jour>
                       </jour>
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Friday, 7th September
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<jour nb="7">
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Kill :
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                      <date>Friday, September 7th 2012</date>
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- Result of the transformation of BS 168 :  
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                      <titre>Kill</titre>
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- all the negative controls plates are empty : the erythromycin concentration is enough high to do a selection.
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<description>
-
- some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to make miniprep and analyse their DNA.
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<ul>
-
- Transformation of BS 168 : new try by electroporation. As the last transformation, the BS 168 strain is transformed by pBK 25, pBK 26, pBK 28, pBK 38 and dispersine in pBK26.
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<li>Result of the transformation of BS 168 : </li>
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Surfactant:
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      <ul>
 +
            <li>All the negative controls plates are clear : the erythromycin concentration is high enough to do a selection;</li>
 +
            <li>Some plates with the transformed bacteria are empty but some have from 1 to 4 clones : these clones are put in liquid cultures in order to make miniprep and analyse their DNA;</li>
 +
      </ul>
 +
<li>Transformation of BS 168 : new trial by electroporation. As the last transformation, the BS 168 strain is transformed by pBK25, pBK26, pBK28, pBK38 and dispersin in pBK26.</li>
 +
</ul>
 +
</description>
 +
                      <titre>Surfactant</titre>
 +
<description>
 +
All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 and pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (sfp, abrB and lacI) would be higher.
 +
</description>
 +
                      </jour>
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All the screened clones had the religated vector, so we decided to use commercial competent cells from STLB2 strain to transform the ligation of pBK39 AND pBK22 in order to diminish the number of clones so that the chances of finding the recombinant plasmid containing the 3 genes (sfp, abrB and lacI) would be higher;
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<jour nb="8">
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Saturday, 8th September
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                      <date>Saturday, September 8th 2012</date>
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Kill :
+
                      <titre>Kill</titre>
-
- Miniprep of the clones BS 168 transformed by pBK26, pBK28 : we use the same protocol than to extract the DNA of E.coli except that we add Lysosyme with the buffer A1. Electrophoresis to analyse the DNA extracted : there is nothing on the gel => Maybe the DNA isn’t concentrated enough ?
+
<description>
 +
Miniprep of the clones BS 168 transformed by pBK26, pBK28 : we use the same protocol as DNA extraction of <i>E.coli</i> with addition of Lysosyme the buffer A1. Electrophoresis to analyze extracted DNA : there is nothing on the gel => Maybe the DNA isn’t concentrated enough ?<br />
New clones are put in liquid cultures in order to make other minipreps.  
New clones are put in liquid cultures in order to make other minipreps.  
-
Surfactant:
+
</description>
 +
                      <titre>Surfactant</titre>
 +
<description>
 +
16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened.
 +
</description>
 +
                      </jour>
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16 clones containing the ligation of pBK22 and pBK39 transformed into STLB2 commercial competent cells are screened;
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<jour nb="9">
-
Sunday, 9th September
+
                      <date>Sunday, September 9th 2012</date>
-
Kill :
+
                      <titre>Kill</titre>
-
- Miniprep of the clones BS 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyse the DNA extracted :
+
<description>
-
- the clone BS 168 transformed by pBK25 has the right plasmid !! =)
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<ul>
-
- the clone BS 168 transformed by pBK28 has the right plasmid !! BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D
+
<li>Miniprep of the clones BS 168 transformed by pBK25, pBK26, pBK28, and Dispersin in pBK26. Electrophoresis to analyze extracted DNA :</li>
-
- the clones BS 168 transformed by pBK26 and dispersine in pBK26, there is nothing on the gel ...  
+
      <ul>
-
 
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            <li>BS 168 clone transformed by pBK25 has the right plasmid !! =)</li>
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- Transformation of BS 168 GFP with the five same plasmids as before. The transformed bacteria are spread on GL + ERY plates, with two different concentrations : [ERY]=10 µg/mL and [ERY]=15 µg/mL.
+
    <li>BS 168 clone transformed by pBK28 has the right plasmid !! BACILLUS LYSOSTAPHIN IS HERE !! :D :D :D</li>
-
- Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method  to concentrate the DNA of the miniprep. We see very light strips on the gel, but the results for pBK26 and dispersine in pBK26 need to be confirm again...
+
    <li>BS 168 clones transformed by pBK26 and dispersine in pBK26 : nothing on the gel ... </li>
-
- Thanks to the good results, we put in storage the transformed Bacillus under the reference :
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    </ul>
-
- BK41 : BS 168 + pBK25
+
<li>Transformation of BS 168 GFP with the same five plasmids as before. The transformed bacteria are spread on GL + ERY plates, with two different concentrations : [ERY]=10 µg/mL and [ERY]=15 µg/mL.</li>
-
- BK42 : BS 168 + pBK28
+
<li>Given the fact that there was nothing on the miniprep for pBK26 and pBK28, we made a new electrophoresis after using the seeDNA method  to concentrate the DNA of the miniprep. We see very light stripes on the gel, but the results for pBK26 and dispersin in pBK26 have to be confirm again...</li>
-
- BK43 : BS 168 + pBK26 (to confirm)
+
<li>Thanks to the good results, we put in storage the transformed Bacillus under the reference :</li>
-
- BK44 : BS 168 + Disp in pBK26 (to confirm)
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      <ul>
-
Surfactant:
+
            <li>BK41 : BS 168 + pBK25</li>
-
 
+
            <li>BK42 : BS 168 + pBK28</li>
-
the electrophoresis of the 16 digested plasmids extracted does not turn out as expected: all clones are in fact the initial plasmid, pBK39!
+
            <li>BK43 : BS 168 + pBK26 (to confirm)</li>
 +
            <li>BK44 : BS 168 + Disp in pBK26 (to confirm)</li>
 +
      </ul>
 +
</ul>
 +
</description>
 +
                      <titre>Surfactant</titre>
 +
<description>
 +
The electrophoresis of the 16 digested plasmids extracted did not turn out as expected : all clones are in fact the initial plasmid, pBK39!
 +
</description>
 +
                      </jour>

Revision as of 00:54, 18 September 2012


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