Team:Grenoble-EMSE-LSU/Documentation/Notebook/August

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<h1>August</h1>
<h1>August</h1>
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                                            <h2>Week 1</h2>
 
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<div id="box" style="width: 700px; margin-left: 137px; padding: 5px; border: 3px solid #000; background-color: #fe2b33;">
 
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<div id="template" style="text-align: center; font-weight: bold; font-size: large; color: #f6f6f6; padding: 5px;">
 
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READ THESE INSTRUCTIONS.
 
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</div>
 
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<div id="instructions" style="text-align: center; font-weight: normal; font-size: small; color: #f6f6f6; padding: 5px;">
 
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Nicolas Roehri is currently working on this page. <strong>DO NOT EDIT THIS PAGE!!!</strong> </br>
 
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</div>
 
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</div>
 
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                                            <h2>Week 1</h2>
                                           <h3>Thursday</h3>
                                           <h3>Thursday</h3>
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    <p>- PCR of mRFP and KR for pJT106b with EcoRI restriction sites on both primers</br>
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    <p></p>
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- Try to digest mRFP and KR with EcoRI but forget pJT106b so there is no control, don’t know if the digestion was efficient or not.</br></p>
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                                           <h3>Friday</h3>
                                           <h3>Friday</h3>
    <p></p>
    <p></p>
                                         <h2>Week 2</h2>
                                         <h2>Week 2</h2>
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                                          <p>- Have some trouble with the digestion of pJT106b. The restriction enzyme used - EcoRI - seems to have a star-activity and cut the plasmid everywhere.</br>
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After these 3 experiences it can be said that the problem comes from the plasmid since the buffers and the RE work on and another plasmid.</br>
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After talking about it with the advisors, they said that during the Miniprep, it still may be some ethanol after the elution. And ethanol can led to the inhibition or the star-activity of the RE.</br>
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To avoid this, before the elution, the column is warm up to 70°C for 5min in order for the ethanol to evaporate.</br>
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</p>
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<p><strong>pBad RBS sspB</strong><br>
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miniprep on the biobrick pBad. PCR of RBS-sspB and PCR clean-up result : 12,8ng/µL</p>
                                           <h3>Monday</h3>
                                           <h3>Monday</h3>
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    <p></p>
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    <p>Starting the construction about <a href="https://static.igem.org/mediawiki/2013/7/73/Grenoble_PCR_of_Killer_Red_ssrA.pdf">KR tagging with ssrA</a>.
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PCR with primers 3’-right KRssRa and 5’-left KR
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</p>
                                           <h3>Tuesday</h3>
                                           <h3>Tuesday</h3>
    <p></p>
    <p></p>
                                           <h3>Wednesday</h3>
                                           <h3>Wednesday</h3>
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    <p></p>
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    <p>Restriction of the PCR product KRssrA</p>
                                           <h3>Thursday</h3>
                                           <h3>Thursday</h3>
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    <p></p>
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    <p>Ligation of KRssrA with pQE30 and transfection</p>
                                           <h3>Friday</h3>
                                           <h3>Friday</h3>
    <p></p>
    <p></p>
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<h3>Saturday</h3>
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    <p>Transfection seems to be good. Verification by purification and PCR and restriction enzyme. Transfection is a fail.</p>
                                             <h2>Week 3</h2>
                                             <h2>Week 3</h2>
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<p>- Redo the PCR for pJT106b with mRFP and KR, and do another miniprep with the new protocol to extract pJT106b.</br>
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- Try again the digestion with lower amount of DNA to be sure that it works to avoid losing to much DNA. it works for different different gel but another problem was encountered. After the gel extraction, done to purified the vector the amount of DNA was too low (<2ng/µL) to do the next experiences. So different wells were use for one column but it was  still not enough (<5ng/µL for 50µL). Try to precipitate the DNA in a fewer volume but it didn’t reach the expected goal.</br>
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10 wells were used for 1 column, and the concentration that we got was of 16.6ng/µL for 30µL. It is not a lot but it is enough for doing the dephosphorylation - to prevent the plasmid to recircularise.</br></p>
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<p>Test to understand why the KRssrA construction has failed.</p>
                                               <h3>Monday</h3>
                                               <h3>Monday</h3>
                                               <h3>Tuesday</h3>
                                               <h3>Tuesday</h3>
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<p> According to the microscopy acquisitions, Diaminofluorescein and Mitotracker Green FM do not seem to cross cell membranes. One explanation could be that the loading dye has time to leave the cell cytoplasm during the wash, between the end of the incubation and the beginning of the observation. It has to be seen if this wash process can be skipped to save time and thus increase the probability of visualizing stained cells under the microscope. </p>
<p> According to the microscopy acquisitions, Diaminofluorescein and Mitotracker Green FM do not seem to cross cell membranes. One explanation could be that the loading dye has time to leave the cell cytoplasm during the wash, between the end of the incubation and the beginning of the observation. It has to be seen if this wash process can be skipped to save time and thus increase the probability of visualizing stained cells under the microscope. </p>
</br>
</br>
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<p> 2) Performed a new kinetics on M15[pRep4-pQE30::KR] cells, in M9 medium. </p>
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<p> 2) Performed a new kinetics on M15[pRep4-pQE30::KR] cells, in <a href="https://static.igem.org/mediawiki/2013/5/50/Grenoble_Preparation_of_M9_medium.pdf">M9 medium</a>. </p>
                                                     <h5> Materials and Methods </h5>
                                                     <h5> Materials and Methods </h5>
<p>
<p>
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<p></p>
<p></p>
                                               <h3>Friday</h3>
                                               <h3>Friday</h3>
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<p><strong>pBad-RBS-sspB</strong>
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                                             <h2>Week 4</h2>
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<br>reculture of the BioBrick sspB <a href="http://parts.igem.org/Part:BBa_K174000"> BBa_K174000</a> to perform new PCR<br> Digestion of pBad <a href="http://parts.igem.org/Part:BBa_K206000"> BBa_206000</a></p>                 
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                                             <h2>Week 4</h2><p>Restart KRssrA construction, PCR. Fail, problems with reagents. Try the construction with Pfu.</p>
                                               <h3>Monday</h3>
                                               <h3>Monday</h3>
                                               <h3>Tuesday</h3>
                                               <h3>Tuesday</h3>
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                                               <h3>Thursday</h3>
                                               <h3>Thursday</h3>
                                               <h3>Friday</h3>
                                               <h3>Friday</h3>
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<p> Digestion with restriction enzymes of PCR Pfu product</p>
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<p>
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</p>
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<p><strong>pBad-RBS-sspB</strong>
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<br>Digestion of RBS-sspB. Ligation of pBad and RBS-sspB. Test digestion and gel migration
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<p align="center"><img src="https://static.igem.org/mediawiki/2013/f/f3/Grenoble_Sarah_digest_2308.png" width="450px"></p>
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<p id="legend">The seven firsts wells are pBad-RBS-sspB digested with ''EcoRI'' the last one is pBad digested with ''EcoRI''</p>
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Latest revision as of 01:28, 5 October 2013

Grenoble-EMSE-LSU, iGEM


Grenoble-EMSE-LSU, iGEM

Retrieved from "http://2013.igem.org/Team:Grenoble-EMSE-LSU/Documentation/Notebook/August"