Difference between revisions of "GDSYZX-United/PROJECT/preparation arabidopsis protoplast"

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<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/project_description">Project description</a></li>
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<li><a href="https://2016.igem.org/Team:GDSYZX-United/Description">Project description</a></li>
 
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/extract _dna">Extract DNA in arabidopsis thaliana</a></li>
 
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/extract _dna">Extract DNA in arabidopsis thaliana</a></li>
 
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/enzyme_cleavage">Enzyme cleavage</a></li>
 
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/enzyme_cleavage">Enzyme cleavage</a></li>
 
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/preparation_arabidopsis_protoplast">Preparation of the Arabidopsis protoplast</a></li>
 
<li><a href="https://2016.igem.org/GDSYZX-United/PROJECT/preparation_arabidopsis_protoplast">Preparation of the Arabidopsis protoplast</a></li>
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<p><div class="text_font"><strong>Place:</strong>Biology Experiment Teaching Center of Sun Yat-sen University</div></p>
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<p><div class="text_font" style="text-indent:0em;"><strong>Place:</strong>Biology Experiment Teaching Center of Sun Yat-sen University</div></p>
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<p><div class="text_font"><strong>Date:</strong>July 28th, 2016</div></p>
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<p><div class="text_font"><strong>Experimental apparatus:</strong>Shaking table, 50 ml centrifuge tube, filtrating equipment, analytical balance, measuring cylinder</div></p>
 
<p><div class="text_font"><strong>Experimental apparatus:</strong>Shaking table, 50 ml centrifuge tube, filtrating equipment, analytical balance, measuring cylinder</div></p>
 
<p><div class="text_font"><strong>Experimental drugs:</strong>Cellulase hydrolysate, W5 solution</div></p>
 
<p><div class="text_font"><strong>Experimental drugs:</strong>Cellulase hydrolysate, W5 solution</div></p>
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<p><div class="text_font"><strong>Procedure:</strong></div></p>
 
<p><div class="text_font"><strong>Procedure:</strong></div></p>
 
<ol>
 
<ol>
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<p><div class="text_font"><li>
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<p><div class="text_font"><li style="text-indent:0em;">
 
the configuration of Cellulase hydrolysate<br>
 
the configuration of Cellulase hydrolysate<br>
 
<table class="table">
 
<table class="table">
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</table>
 
</table>
 
</li></div></p>
 
</li></div></p>
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<p><div class="text_font"><li>
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<p><div class="text_font"><li style="text-indent:0em;">
 
Pick Arabidopsis leaves and cut out them with 0.5-1mm, then immerse it in 5ml  Cellulase hydrolysate.
 
Pick Arabidopsis leaves and cut out them with 0.5-1mm, then immerse it in 5ml  Cellulase hydrolysate.
 
</li></div></p>
 
</li></div></p>
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<p><div class="text_font"><li>
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<p><div class="text_font"><li style="text-indent:0em;">
 
Put them in shaking table for 30min in the dark, then waiting for another 3h without shaking. When it comes to green and then gently shake it.
 
Put them in shaking table for 30min in the dark, then waiting for another 3h without shaking. When it comes to green and then gently shake it.
 
</li></div></p>
 
</li></div></p>
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<p><div class="text_font"><li>
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<p><div class="text_font"><li style="text-indent:0em;">
 
(Adding 5ml W5 solution and then filtrating.)
 
(Adding 5ml W5 solution and then filtrating.)
 
</li></div></p>
 
</li></div></p>
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<p><div class="text_font"><li>
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<p><div class="text_font"><li style="text-indent:0em;">
 
The protoplast was observed under the microscope
 
The protoplast was observed under the microscope
 
</li></div></p>
 
</li></div></p>
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<div class="col-sm-2"></div>
 
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<p><div class="text_font"><strong>Result:</strong></div></p>
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<p><div class="text_font">There is no protoplast in Cellulase hydrolysate (it didn’t come to green), and the cell wall of young leaves were not damaged.</div></p>
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<p><div class="text_font"><strong>Introspection:</strong></div></p>
 
<p><div class="text_font"><strong>Introspection:</strong></div></p>
 
<p><div class="text_font">
 
<p><div class="text_font">
 
<ol>
 
<ol>
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<li>Cutting leaves without using the blade but by hand , or deal it with a scalpel pulling, which may damaged the cells.</li>
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<li style="text-indent:0em;">Cutting leaves without using the blade but by hand , or deal it with a scalpel pulling, which may damaged the cells.</li>
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<li>The shaking table should not exceed 80r/min.</li>
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<li style="text-indent:0em;">The shaking table should not exceed 80r/min.</li>
 
</ol>
 
</ol>
 
</div></p>
 
</div></p>
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<div class="col-sm-8">
 
<div class="col-sm-8">
 
<div align="center">
 
<div align="center">
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<img alt="image" class="img-responsive" src="img/project_prepration1.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/a/a6/T--GDSYZX-United--project_prepration1.jpg">
 
<p><div class="text_font">Filtrate during the configuration of Cellulase hydrolysate</div></p>
 
<p><div class="text_font">Filtrate during the configuration of Cellulase hydrolysate</div></p>
 
</div>
 
</div>
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<div class="col-sm-4">
 
<div align="center">
 
<div align="center">
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<img alt="image" class="img-responsive" src="img/project_prepration2.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/d/da/T--GDSYZX-United--project_prepration2.jpg">
 
<p><div class="text_font">Cut out the leaves</div></p>
 
<p><div class="text_font">Cut out the leaves</div></p>
 
</div>
 
</div>
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<div class="col-sm-4">
 
<div align="center">
 
<div align="center">
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<img alt="image" class="img-responsive" src="img/project_prepration3.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/c/cf/T--GDSYZX-United--project_prepration3.jpg">
 
<p><div class="text_font">Immerse Arabidopsis leaves in 5ml  Cellulase hydrolysate.</div></p>
 
<p><div class="text_font">Immerse Arabidopsis leaves in 5ml  Cellulase hydrolysate.</div></p>
 
</div>
 
</div>
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<div class="col-sm-8">
 
<div align="center">
 
<div align="center">
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<img alt="image" class="img-responsive" src="img/project_prepration4.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/e/e8/T--GDSYZX-United--project_prepration4.jpg">
 
<p><div class="text_font">Prepare the microscopic slides </div></p>
 
<p><div class="text_font">Prepare the microscopic slides </div></p>
 
</div>
 
</div>
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<div class="col-sm-6">
 
<div class="col-sm-6">
 
<div align="center">
 
<div align="center">
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<img alt="image" class="img-responsive" src="img/project_prepration5.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/2/2f/T--GDSYZX-United--project_prepration5.jpg">
 
<p><div class="text_font">Cellulase hydrolysate under microscope (nothing).</div></p>
 
<p><div class="text_font">Cellulase hydrolysate under microscope (nothing).</div></p>
 
</div>
 
</div>
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<div class="col-sm-6">
 
<div class="col-sm-6">
 
<div align="center">
 
<div align="center">
−
<img alt="image" class="img-responsive" src="img/project_prepration6.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/c/c8/T--GDSYZX-United--project_prepration6.jpg">
 
<p><div class="text_font">Plant cells under microscope.</div></p>
 
<p><div class="text_font">Plant cells under microscope.</div></p>
 
</div>
 
</div>

Latest revision as of 14:53, 17 October 2016

Preparation of the Arabidopsis protoplast

Place:Biology Experiment Teaching Center of Sun Yat-sen University

Experimental apparatus:Shaking table, 50 ml centrifuge tube, filtrating equipment, analytical balance, measuring cylinder

Experimental drugs:Cellulase hydrolysate, W5 solution

Procedure:

  1. the configuration of Cellulase hydrolysate
    Cellulase 0.225g
    Mecerozyme R10 0.045g
    mannitol 1.09g
    KCl 1.5g
    MES 4.18g
    water 12ml
    cooled down to room temperature after waiting in 55℃ water 10min.
    CaCl2 1.11g
    BSA 1ml
    water 2ml
    filtrating

  2. Pick Arabidopsis leaves and cut out them with 0.5-1mm, then immerse it in 5ml Cellulase hydrolysate.

  3. Put them in shaking table for 30min in the dark, then waiting for another 3h without shaking. When it comes to green and then gently shake it.

  4. (Adding 5ml W5 solution and then filtrating.)

  5. The protoplast was observed under the microscope

Introspection:

  1. Cutting leaves without using the blade but by hand , or deal it with a scalpel pulling, which may damaged the cells.
  2. The shaking table should not exceed 80r/min.

image

Filtrate during the configuration of Cellulase hydrolysate

image

Cut out the leaves

image

Immerse Arabidopsis leaves in 5ml Cellulase hydrolysate.

image

Prepare the microscopic slides

image

Cellulase hydrolysate under microscope (nothing).

image

Plant cells under microscope.