Team:Concordia/Notebook

From 2013.igem.org

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     <div class="wrapper"><div class="site-menu col-md-2"><div id="p-logo">
     <div class="wrapper"><div class="site-menu col-md-2"><div id="p-logo">
   <a title="Main Page" href="/Main_Page">
   <a title="Main Page" href="/Main_Page">
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     <img src="https://static.igem.org/mediawiki/2013/0/08/Igem_logo.png">
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     <img src="https://static.igem.org/mediawiki/2013/0/02/TB_IGEM_official_logo.png">
   </a>
   </a>
</div>  
</div>  
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     <li><a href="https://2013.igem.org/Team:Concordia">Home</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia">Home</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Project">Project</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Project">Project</a></li>
 +
      <ul class="project-list">
 +
          <li><a href="https://2013.igem.org/Team:Concordia/GasClock">Gas Clock</a></li>
 +
          <li><a href="https://2013.igem.org/Team:Concordia/Logic">Logic</a></li>
 +
          <li><a href="https://2013.igem.org/Team:Concordia/Interface">Interface</a></li>
 +
          <li><a href="https://2013.igem.org/Team:Concordia/Memory">Memory</a></li>
 +
      </ul>
     <li><a href="https://2013.igem.org/Team:Concordia/Team">Team</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Team">Team</a></li>
     <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Concordia">Official Profile</a></li>
     <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Concordia">Official Profile</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Parts">Registry</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Parts">Registry</a></li>
 +
    <li><a href="https://2013.igem.org/Team:Concordia/HumanPractices">Human Practices</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Modeling">Modeling</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Modeling">Modeling</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Notebook">Notebook</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Notebook">Notebook</a></li>
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   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Failure for PCR amplification of Ethylene Cassette synthesis genes.  </h2>
+
       <h2 class="log-title">Gene Regulatory Network Completed</h2>
-
       <h4 class="log-time">Sept, 13th</h4>
+
       <h4 class="log-time">June, 27th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/c/c9/Sept_13.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/74/June_27.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Yeast cell strain, for yeast assembly of the ethylene cassette genes, was made and thus needed its parts to assemble. The amplification of ethylene cassette genes had been previously attempted and failed. This same PCR was then attempted again on a different PCR machine and using a temperature gradient from 52 degrees to 60 degrees Celsius; the results was still unsuccessful. Upon review all reverse primers used for said genes were incorrect; this is one definite source of error!</p>
+
       <p class="log-long-desc">After 2 months of meetings, discussion and research we have finalized our genetic regulatory network. Next stop… wet lab!</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">GFP amplified in mini-prep samples</h2>
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       <h2 class="log-title">Amplification of ETR1 and EnvZ Fragments Successful</h2>
-
       <h4 class="log-time">Sept, 12th</h4>
+
       <h4 class="log-time">July, 26th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/7c/Sept_12.PNG"/>
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       <img class="log-img" src="https://static.igem.org/mediawiki/2013/3/32/July_26.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples.</p>
+
       <p class="log-long-desc">We successfully amplified truncated fragments of ETR1 and EnvZ from A. thaliana cDNA (provided by Patrick Gulick, PhD) and E. coli genomic DNA (provided by Michelle Harvey, Tech). These truncated fragments will be used to generate site specific fusion proteins.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Mini-Prep of PompC transformed E.coli cells. </h2>
+
       <h2 class="log-title">Generated Fusions</h2>
-
      <h4 class="log-time">Sept, 10th</h4>
+
       <h4 class="log-time">July, 29th</h4>
-
    </div>
+
-
   
+
-
    <div class="log-desc">
+
-
      <p class="log-long-desc">Although all cells appeared red in color, all samples (10 in total) were mini-preped according to kit procedures. </p>
+
-
    </div>
+
-
  </div>
+
-
 
+
-
  <div class="log col-md-9 log-left">
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-
    <div class="log-title-wrap">
+
-
      <h2 class="log-title">Overlapping PCR of RBS/Plac and Fusions was unsuccessful</h2>
+
-
       <h4 class="log-time">Sept, 9th</h4>
+
-
    </div>
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-
   
+
-
    <div class="log-img-wrap">
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-
      <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/7d/Sept_9_1.PNG"/>
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     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/1/17/Sept_9_2.PNG"/>
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       <img class="log-img" src="https://static.igem.org/mediawiki/2013/0/01/July_29.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.</p>
+
       <p class="log-long-desc">We successfully extracted the amplicons (from 07-26) from 1% Agarose gel to be used in subsequent overlapping PCR experiments. This was a fundamental step as any residual primers within the sample will make it difficult to perform the overlapping PCR.</p>
-
    </div>
+
-
  </div>
+
-
 
+
-
  <div class="log col-md-9 log-right">
+
-
    <div class="log-title-wrap">
+
-
      <h2 class="log-title">Overlapping PCR of RBS/Plac and Fusions was unsuccessful</h2>
+
-
      <h4 class="log-time">Sept, 10th</h4>
+
-
    </div>
+
-
   
+
-
    <div class="log-desc">
+
-
      <p class="log-long-desc">Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.</p>
+
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.</h2>
+
       <h2 class="log-title">Generated Fusions</h2>
-
       <h4 class="log-time">Sept, 3rd</h4>
+
       <h4 class="log-time">Aug, 1st</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/2/28/Sept_3.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/c/cd/Aug_1.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.</p>
+
       <p class="log-long-desc">Overlapping PCR was attempted using a troubleshooting technique. Fragments used came from E.coli (EnvZ) and A. thaliana (ETR1). One nanogram of ETR1 and EnvZ fragments, corresponding to their proper fusion domains (DOM – domain, CON- conserved, and LIT-literature), were loaded using the standard contents necessary for PCR minus the primers. Four differing DNA polymerases were tested; PhireHot Start Polymerase, Phusion polymerase, Crimson Taq polymerase, and Q5 polymerase.  Primers were added after 10 cycles out of a total of 30 cycles. PCR samples were then verified using a 1% agarose gel containing EtBr, and tested against ETR1 and EnvZ fragments (positive controls). Electrophoresis was conducted for 50 minutes at 100volts. Visualization revealed that out of the four polymerases used, only one was able to generated the correctly sized fusion proteins; Phire Hot Start DNA polymerase.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Ethanol Purification of ETR1, PLac, and PompC-GFP</h2>
+
       <h2 class="log-title">Amplified EnvZ, but No LIT fusion</h2>
-
       <h4 class="log-time">Aug, 22nd</h4>
+
       <h4 class="log-time">Aug, 6th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/4/47/Aug_22.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/a/ac/Aug_6.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">ETR1, PLac and PompC-GFP were Ethanol Purified.</p>
+
       <p class="log-long-desc">Full length ETR1 and EnvZ fragments were needed for Nested Deletion protocol, also the LIT fusion protein required increased yield. Amplification PCR of all of these samples was conducted. Standard PCR components were loaded, including correct primers, with 1 ng of genomic DNA (for EnvZ), 3ng of A. thaliana cDNA (for ETR1), and 1 ng of previously generated LIT fusion. PCR samples were run for 30 cycles, and then tested via Gel Electrophoresis, using a 1% agarose gel containing EtBr, at 100 volts for 50 minutes.  For out Nested Deletions samples; ENVz was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Also, our LIT fusion amplification, using DNA template from the previously successful overlapping PCR, was not achieved.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP. </h2>
+
       <h2 class="log-title">Ethanol purification of EnvZ, and Fusions DOM/CON</h2>
-
       <h4 class="log-time">Aug, 21st</h4>
+
       <h4 class="log-time">Aug, 7th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/5/51/Aug_21.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/e/ec/Aug_7.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!</p>
+
       <p class="log-long-desc">Any further purification of samples was suggested to be done using an ethanol purification protocol. Previously amplified Nested Deletion EnvZ, and Fusions proteins DOM and CON were Ethanol purified. Ethanol purification was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. Ethanol Purification products were then verified and quantified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Concentrations, determined through gel quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion. Additionally, amplification PCR for ETR1 and LIT Fusion were attempted again but were unsuccessful.</p>
     </div>
     </div>
   </div>
   </div>
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     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">PCR amplification of interface genes was commenced; RhlR was successfully amplified and purified. RhlR was quantified to be 360 ng/ul. Gas clock component 'TetR' was also successfully amplified, and later purified. TetR quantification yield was 216 ng/ul. Additionally, PompC-GFP was successfully ethanol purified and quantified to be approximately 270 ng/ul.   </p>
+
       <p class="log-long-desc">PCR amplification of; interface gene RhlR, and repressor protein TetR was conducted. Standard PCR components, including specific and correct primers, were added to 200-600 pg of target DNA. Ethanol Purification of RhlR, TetR, and PompC-GFP was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. Products were then verified and quantified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Concentrations, determined through gel quantification, are estimated as; 360 ng/ul for RhlR, 216 ng/ul for TetR, and 270 ng/ul for PompC-GFP.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Phire Discontinued</h2>
+
       <h2 class="log-title">Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP. </h2>
-
       <h4 class="log-time">Aug, 12th</h4>
+
       <h4 class="log-time">Aug, 21st</h4>
 +
    </div>
 +
   
 +
    <div class="log-img-wrap">
 +
      <img class="log-img" src="https://static.igem.org/mediawiki/2013/5/51/Aug_21.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">The Phire DNA polymerase sample we used was obtained from the Dr.Martin lab. Since it was only a trial sample, more Phire DNA polymerase needed to be ordered. We absolutely wanted to order Phire DNA polymerase since it had proven to be successful previously (Aug 01). But when we attempted to order it we figured out that the original Phire, and its second generation 'Phire Hot start Green polymerase' had been discontinued.</p>
+
       <p class="log-long-desc">Temperature Gradient Amplification PCR was attempted for the ETR1 and Literature Fusion, since they were proving difficult to amplify. PCR was conduct using standard components, including specific primers, and 1 ng of Lit Fusion DNA, and 3ng of ETR1 cDNA. A positive control of EnvZ was also run in order to rule out potential discrepancies. The temperature gradient was set from 58 degrees Celsius to 62 degrees Celsius, and number of cycles was 35. Additionally, normal amplification PCR was conducted for 200-600 pg of Plac, and 1 ng of PompC-GFP DNA using standard protocol. Products were then tested using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization of the temperature gradient revealed that ETR1 products were amplified at the correct size for temperatures 60 to 63, and Lit fusion products were amplified correctly at 62 and 64. Also, Plac and PompC-GFP were also amplified properly.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Ethanol purification of EnvZ, and Fusions DOM/CON</h2>
+
       <h2 class="log-title">Ethanol Purification of ETR1, PLac, and PompC-GFP</h2>
-
       <h4 class="log-time">Aug, 7th</h4>
+
       <h4 class="log-time">Aug, 22nd</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/e/ec/Aug_7.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/4/47/Aug_22.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Amplification of ETR1 and LIT Fusion were attempted again but were unsuccessful. On the other hand, the Ethanol Purification of EnvZ, and Fusions DOM and CON were carried out successfully! Concentrations, via Gel Quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion.</p>
+
       <p class="log-long-desc">Previously amplified ETR1, PLac and PompC-GFP were Ethanol purified. Ethanol purification was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water.  Ethanol Purification products were then verified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization shows that ETR1 was purified at various concentrations depending on which sample was used (where 61 degrees was the best), PLac and PompC-GFP were also purified accurately.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Amplified EnvZ, but No LIT fusion</h2>
+
       <h2 class="log-title">Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.</h2>
-
       <h4 class="log-time">Aug, 6th</h4>
+
       <h4 class="log-time">Sept, 3rd</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/a/ac/Aug_6.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/2/28/Sept_3.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.</p>
+
       <p class="log-long-desc">Troubleshooting PCR amplification of; interface genes RhlR, LuxR, and LuxI was conducted. RhlI was used as a positive control since its amplification had been successful in numerous other PCR reactions. Standard PCR components, including specific and correct primers, were added to 200-600 pg of target DNA. PCR reactions tested common troubleshooting techniques such as; reduced primers, DMSO, added Magnesium content, and Taq polymerase. Ethanol Purification of DOM, CON and LIT fusions as well as Plac was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. PCR products were then verified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Out of the four parameters tested (plus one normal reaction); only added magnesium gave a visible result for LuxI and Lux R. However, the bands obtained were weak, therefore this reaction must be attempted again.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Low Purification Yield; Fusions</h2>
+
       <h2 class="log-title">Mini-Prep of PompC transformed E.coli cells. </h2>
-
       <h4 class="log-time">Aug, 2nd</h4>
+
       <h4 class="log-time">Sept, 10th</h4>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Our Fusions that were generated using 'Phire DNA polymerase' were Gel Purified and visualized using Gel Electrophoresis. What we saw was not good news, Fusions LIT and CON were not concentrated enough. DOM, and CON were usable but LIT was not. LIT fusion has to be amplified.</p>
+
       <p class="log-long-desc">All cells appeared red in color after transformation, but mini prep was still attempted. All samples (10 in total) were mini-preped according to kit procedures.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Generated Fusions</h2>
+
       <h2 class="log-title">GFP amplified in mini-prep samples</h2>
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       <h4 class="log-time">Aug, 1st</h4>
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       <h4 class="log-time">Sept, 12th</h4>
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       <img class="log-img" src="https://static.igem.org/mediawiki/2013/c/cd/Aug_1.PNG"/>
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       <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/7c/Sept_12.PNG"/>
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     <div class="log-desc">
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       <p class="log-long-desc">Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!</p>
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       <p class="log-long-desc">Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tested for PompC using restriction digest and PCR amplification. Standard PCR components, including specific and correct primers, were added to ~1ng of target DNA. Restriction Digest was conducted using standard Biobricks cut sites. PCR products and Restriction digest products, were visualized using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization revealed that PCR samples yielded a positive result for PompC-GFP in mini-preped samples, while restriction digest products also cut out a fragment the same size as PompC-GFP.</p>
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Latest revision as of 02:48, 28 September 2013

Notebook

Gene Regulatory Network Completed

June, 27th

After 2 months of meetings, discussion and research we have finalized our genetic regulatory network. Next stop… wet lab!

Amplification of ETR1 and EnvZ Fragments Successful

July, 26th

We successfully amplified truncated fragments of ETR1 and EnvZ from A. thaliana cDNA (provided by Patrick Gulick, PhD) and E. coli genomic DNA (provided by Michelle Harvey, Tech). These truncated fragments will be used to generate site specific fusion proteins.

Generated Fusions

July, 29th

We successfully extracted the amplicons (from 07-26) from 1% Agarose gel to be used in subsequent overlapping PCR experiments. This was a fundamental step as any residual primers within the sample will make it difficult to perform the overlapping PCR.

Generated Fusions

Aug, 1st

Overlapping PCR was attempted using a troubleshooting technique. Fragments used came from E.coli (EnvZ) and A. thaliana (ETR1). One nanogram of ETR1 and EnvZ fragments, corresponding to their proper fusion domains (DOM – domain, CON- conserved, and LIT-literature), were loaded using the standard contents necessary for PCR minus the primers. Four differing DNA polymerases were tested; PhireHot Start Polymerase, Phusion polymerase, Crimson Taq polymerase, and Q5 polymerase. Primers were added after 10 cycles out of a total of 30 cycles. PCR samples were then verified using a 1% agarose gel containing EtBr, and tested against ETR1 and EnvZ fragments (positive controls). Electrophoresis was conducted for 50 minutes at 100volts. Visualization revealed that out of the four polymerases used, only one was able to generated the correctly sized fusion proteins; Phire Hot Start DNA polymerase.

Amplified EnvZ, but No LIT fusion

Aug, 6th

Full length ETR1 and EnvZ fragments were needed for Nested Deletion protocol, also the LIT fusion protein required increased yield. Amplification PCR of all of these samples was conducted. Standard PCR components were loaded, including correct primers, with 1 ng of genomic DNA (for EnvZ), 3ng of A. thaliana cDNA (for ETR1), and 1 ng of previously generated LIT fusion. PCR samples were run for 30 cycles, and then tested via Gel Electrophoresis, using a 1% agarose gel containing EtBr, at 100 volts for 50 minutes. For out Nested Deletions samples; ENVz was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Also, our LIT fusion amplification, using DNA template from the previously successful overlapping PCR, was not achieved.

Ethanol purification of EnvZ, and Fusions DOM/CON

Aug, 7th

Any further purification of samples was suggested to be done using an ethanol purification protocol. Previously amplified Nested Deletion EnvZ, and Fusions proteins DOM and CON were Ethanol purified. Ethanol purification was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. Ethanol Purification products were then verified and quantified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Concentrations, determined through gel quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion. Additionally, amplification PCR for ETR1 and LIT Fusion were attempted again but were unsuccessful.

Amplification/Ethanol Purification of TetR and RhlR. Ethanol Purification of PompC-GFP

Aug, 13th

PCR amplification of; interface gene RhlR, and repressor protein TetR was conducted. Standard PCR components, including specific and correct primers, were added to 200-600 pg of target DNA. Ethanol Purification of RhlR, TetR, and PompC-GFP was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. Products were then verified and quantified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Concentrations, determined through gel quantification, are estimated as; 360 ng/ul for RhlR, 216 ng/ul for TetR, and 270 ng/ul for PompC-GFP.

Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP.

Aug, 21st

Temperature Gradient Amplification PCR was attempted for the ETR1 and Literature Fusion, since they were proving difficult to amplify. PCR was conduct using standard components, including specific primers, and 1 ng of Lit Fusion DNA, and 3ng of ETR1 cDNA. A positive control of EnvZ was also run in order to rule out potential discrepancies. The temperature gradient was set from 58 degrees Celsius to 62 degrees Celsius, and number of cycles was 35. Additionally, normal amplification PCR was conducted for 200-600 pg of Plac, and 1 ng of PompC-GFP DNA using standard protocol. Products were then tested using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization of the temperature gradient revealed that ETR1 products were amplified at the correct size for temperatures 60 to 63, and Lit fusion products were amplified correctly at 62 and 64. Also, Plac and PompC-GFP were also amplified properly.

Ethanol Purification of ETR1, PLac, and PompC-GFP

Aug, 22nd

Previously amplified ETR1, PLac and PompC-GFP were Ethanol purified. Ethanol purification was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. Ethanol Purification products were then verified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization shows that ETR1 was purified at various concentrations depending on which sample was used (where 61 degrees was the best), PLac and PompC-GFP were also purified accurately.

Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.

Sept, 3rd

Troubleshooting PCR amplification of; interface genes RhlR, LuxR, and LuxI was conducted. RhlI was used as a positive control since its amplification had been successful in numerous other PCR reactions. Standard PCR components, including specific and correct primers, were added to 200-600 pg of target DNA. PCR reactions tested common troubleshooting techniques such as; reduced primers, DMSO, added Magnesium content, and Taq polymerase. Ethanol Purification of DOM, CON and LIT fusions as well as Plac was conducted, using all of the remaining sample, which was precipitated using sodium acetate and ice cold 99% ethanol, followed by 70% ethanol and re-suspension in Ultra Pure Distilled water. PCR products were then verified using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Out of the four parameters tested (plus one normal reaction); only added magnesium gave a visible result for LuxI and Lux R. However, the bands obtained were weak, therefore this reaction must be attempted again.

Mini-Prep of PompC transformed E.coli cells.

Sept, 10th

All cells appeared red in color after transformation, but mini prep was still attempted. All samples (10 in total) were mini-preped according to kit procedures.

GFP amplified in mini-prep samples

Sept, 12th

Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tested for PompC using restriction digest and PCR amplification. Standard PCR components, including specific and correct primers, were added to ~1ng of target DNA. Restriction Digest was conducted using standard Biobricks cut sites. PCR products and Restriction digest products, were visualized using a 1% agarose gel containing EtBr run at 100 volts for 50 min. Visualization revealed that PCR samples yielded a positive result for PompC-GFP in mini-preped samples, while restriction digest products also cut out a fragment the same size as PompC-GFP.