Team:Valencia UPV/Project/modules/methodology

From 2014.igem.org

(Difference between revisions)
(Created page with "{{:Team:Valencia_UPV/header}} <html> <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.11.1/jquery.min.js"></script> <div align="center"><div id...")
Line 23: Line 23:
   
   
         <div id="tab2" class="tab">
         <div id="tab2" class="tab">
-
             <p>/p>
+
             <p></p>
         </div>
         </div>
   
   
         <div id="tab3" class="tab">
         <div id="tab3" class="tab">
              
              
-
             <p></p>
+
             <p class="subpart"></p>
 +
<p>Headspace solid-phase microextraction is one of the most convenient techniques for analysing Volatile Organic Compounds (VOCs). It is a very sensitive, inexpensive, robust and easy-to-use technique since it does not require the use of solvents and is able to detect metabolites up to ppt (parts per trillion) with a low sample quantity.</p><br/><br/>
 +
 
 +
<p>In our case, we chose this technique coupled to a Gas Chromatography-Mass Spectrometry detection (see GC-MS) to analyse the presence of pheromones ((Z)-11 hexadec-1-ol, (Z)-11 hexadecenal, (Z)-11 hexadecenyl acetate)[see biosynthesis] in our samples, Nicotiana benthamiana leaves [see agroinfiltration].</p><br/><br/>
 +
 
 +
<p>This technique is based on the detection of volatiles present in the headspace of a vial. The volatiles diffuse from the sample (Solid phase) to the headspace (Gas phase) and are captured by an absorbent, a polymer-coated fiber with high affinity for them. After desorption from the fiber, volatiles are analysed by GC-MS.</p><br/><br/>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<p>In order to perform an accurate analysis of the sample, this technique must follow 3 simple steps:</p><br/><br/>
 +
 
 +
<ul>
 +
 
 +
<li><a class="black-bold">Sample preparation</a>: it is vital for a proper analysis that samples are unaltered during the whole process.
 +
    <li>Therefore the biological material, leaves in this case, must be immediately kept on liquid Nitrogen after the removal from the plant.</li>
 +
    <li>Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap headspace vial.</li>
 +
    <li>Afterwards, EDTA and saturated solution of CaCl2 are added to inhibit enzymatic activity and stabilize the sample.</li>
 +
    <li>Finally, they are sonicated to induce the release of volatiles from the solid phase to the gas phase in the headspace of the vial.</li>
 +
</li>
 +
 
 +
<li><a class="black-bold">Trapping of volatiles</a>: volatiles present in the gas phase must be extracted for further analysis. Therefore a fiber coated with an adsorbent polymer is introduced in the headspace, and ‘traps’ the volatiles from the sample.</li>
 +
 
 +
<li><a class="black-bold">Desorption</a>: release of the volatiles from the fiber by thermal desorption which takes place in the insertion port of the Gas chromatograph.</li>
 +
 
 +
</ul>
 +
 
 +
<p></p><br/><br/>
 +
 
         </div>
         </div>
   
   

Revision as of 19:07, 12 October 2014


Methodology

Headspace solid-phase microextraction is one of the most convenient techniques for analysing Volatile Organic Compounds (VOCs). It is a very sensitive, inexpensive, robust and easy-to-use technique since it does not require the use of solvents and is able to detect metabolites up to ppt (parts per trillion) with a low sample quantity.



In our case, we chose this technique coupled to a Gas Chromatography-Mass Spectrometry detection (see GC-MS) to analyse the presence of pheromones ((Z)-11 hexadec-1-ol, (Z)-11 hexadecenal, (Z)-11 hexadecenyl acetate)[see biosynthesis] in our samples, Nicotiana benthamiana leaves [see agroinfiltration].



This technique is based on the detection of volatiles present in the headspace of a vial. The volatiles diffuse from the sample (Solid phase) to the headspace (Gas phase) and are captured by an absorbent, a polymer-coated fiber with high affinity for them. After desorption from the fiber, volatiles are analysed by GC-MS.



In order to perform an accurate analysis of the sample, this technique must follow 3 simple steps:



  • Sample preparation: it is vital for a proper analysis that samples are unaltered during the whole process.
  • Therefore the biological material, leaves in this case, must be immediately kept on liquid Nitrogen after the removal from the plant.
  • Then, leaves are ground to a fine powder with mortar and pestle and introduced in a screw cap headspace vial.
  • Afterwards, EDTA and saturated solution of CaCl2 are added to inhibit enzymatic activity and stabilize the sample.
  • Finally, they are sonicated to induce the release of volatiles from the solid phase to the gas phase in the headspace of the vial.
  • Trapping of volatiles: volatiles present in the gas phase must be extracted for further analysis. Therefore a fiber coated with an adsorbent polymer is introduced in the headspace, and ‘traps’ the volatiles from the sample.
  • Desorption: release of the volatiles from the fiber by thermal desorption which takes place in the insertion port of the Gas chromatograph.





Retrieved from "http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology"