Difference between revisions of "Team:Berlin diagnostX/Experiments"

Line 281: Line 281:
 
<!-- Other Experiments -->
 
<!-- Other Experiments -->
  
<!-- Other Experiments -->
 
  
 
<div id="andere_experimente">
 
<div id="andere_experimente">
Line 291: Line 290:
 
<div class="row">
 
<div class="row">
 
     <div class="col-12">
 
     <div class="col-12">
         <h3 class="text-center igem_blue mt-4 mb-1">Other Experiments</h3>
+
         <h4 class="text-center mb-2">Preparation of nucleic acids</h5>
        <h5 class="text-center mb-2">Preparation of nucleic acids</h5>
+
 
         <p class="text-justify">For our experiments, a large set of different DNAs and RNAs - primers, templates, targets and controls – are required in varied concetrations. Thus, a set of standardized but easily adaptable protocols to extract and purify nucleic acids was established.</p>
 
         <p class="text-justify">For our experiments, a large set of different DNAs and RNAs - primers, templates, targets and controls – are required in varied concetrations. Thus, a set of standardized but easily adaptable protocols to extract and purify nucleic acids was established.</p>
 
     </div>
 
     </div>
Line 336: Line 334:
  
  
<!-- Optimizing Screening -->
 
 
<div id="screening_opti">
 
<div class="row">
 
    <div class="col-12">
 
        <h3 class="text-center igem_blue mt-4">Optimizing the Screening Pipeline</h3>
 
    </div>
 
</div>
 
<div class="row mt-4">
 
    <div class="col-6">
 
        <p class="text-justify"><strong>Goal:</strong>Establishing the PureExpress® cell free protein expression kits (PureExpress®, NEB) as our main tool for RNA detection and assess its capabilities.</p>
 
        <p class="text-justify"><strong>Description</strong>The PureExpress®-System is composition of completely recombinant molecules to effectively transcribe any form of DNA into mRNA and then translate this into a protein. To use this system for field diagnostics, it has to meet the following requirements:</p>
 
        <ul>
 
            <li>Heat stability up to a temperature of 50°C</li>
 
            <li>High efficiency even with low volumes of reagents and/or low concentrations</li>
 
            <li>Stability towards changes in electrolyte concentration/ addition of foreign molecules</li>
 
            <li>Long-term storability.</li>
 
        </ul>
 
    </div>
 
    <div class="col-6">
 
        <p class="text-justify">We verified this by evaluating the kinetics of sensor reactions in different experimental conditions, including different temperatures, different reactant concentrations, changes in buffer as well as re-measurements after storage.<br><br>We could show that the system meets all these requirements. Especially noteworthy, we were able to create significant color change in a volume of 2,5 uL which is only 10% of the volume recommended by the manufacturer. Furthermore, we could show that the system is still highly active in concentrations of 40-50%. This makes it possible to use the system with patient samples of only estimated concentration without risking reduced functionality. Last but not least, the system is not vulnerable to possibly interfering molecules, as for example the chlor-phenol-red used  as a reporter dye.
 
        <br> <br> We concluded that the PureExpress® - System is most suitable for our essay; also due to the fact, that it composed of recombinant parts instead of cell lysate and so has very few variability</p>
 
    </div>
 
</div>
 
<div class="row mt-5">
 
    <div class="col-12">
 
        <h5 class="text-center mb-2">Preparation of sensors for iGEM Submission</h5>
 
    </div>
 
</div>
 
<div class="row mt-5">
 
    <div class="col-12">
 
        <h5 class="text-left mb-2 igem_blue">Mutagenesis</h5>
 
        <p class="text-justify"><strong>Goal:</strong>Introduce a silent mutation at position (3181;G>A) in lacZ to remove EcoRI restriction site.</p>
 
        <p class="text-justify"><strong>Description</strong>We used Q5 mutagenesis kit (NEB) and designed primers using NEBaseChanger software. The software recommended a forward-primer introducing the mutation and a reverse primer with suitable tm to complete the plasmid. Since the software neither provided a ranking of other primer pairs nor detailed information on how the primers are chosen, we designed two more primer pairs introducing the mutation. Mutagenesis was carried out according to the manufacturers protocol with 1ng, 8ng and 20ng of plasmid being used for each primer pair. Successful mutagenesis was confirmed by sequencing.</p>
 
    </div>
 
</div>
 
 
<div class="row mt-5">
 
<div class="row mt-5">
 
     <div class="col-12">
 
     <div class="col-12">
         <h5 class="text-left mb-2 igem_blue">Cloning into pSB1C</h5>
+
         <h4 class="text-left mb-2 igem_blue">Cloning into pSB1C</h5>
 
     </div>
 
     </div>
 
</div>
 
</div>

Revision as of 22:15, 1 November 2017

Experiments