Difference between revisions of "Team:Rheda Bielefeld/Methods"

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<b> <a href="https://2018.igem.org/Team:Rheda_Bielefeld/Pollen" style="color:yellow;font-size:25px;"> Pollen </a> </b> <br/> <br/>
 
<b> <a href="https://2018.igem.org/Team:Rheda_Bielefeld/Pollen" style="color:yellow;font-size:25px;"> Pollen </a> </b> <br/> <br/>
  
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For our experiments with pollen, we have used two methods. The first method was using trypsin and a ribolyser. Trypsin is an enzyme mostly found in the digestive system of many animals and can hydrolyze proteins. We have used it here in the hope that the trypsin would hydrolyze the proteins in the pollen´s outer wall and therefore making the pollen break. The Ribolyser is made up from many little balls of ceramics and is used to homogenize biological samples. It works easily: you put a sample into the ribolyser and shake it well, best with a mechanical shaker. By shaking it, the little ceramic balls fly around in the container and by crashing into the sample, a lot of damage can be done to your biological sample. A negative side effect of using the ribolyser is that a lot of heat is produced when the balls fly around. We used the ribolyser to break the pollen walls with both heat and the mechanical damage done. Another way we tried to break open the wall of a pollen was by using liquid nitrogen. When you put liquid nitrogen onto cells, they break because all liquids touching that extremely cold substance freeze and eventually break.<br/> <br/>
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For our experiments with Pollens, we have used two methods. The first method was using trypsin and a ribolyser. Trypsin is an enzyme mostly found in the digestive system of many animals and can hydrolyze proteins. We have used it here in the hope that the trypsin would hydrolyze the proteins in the pollen´s outer wall and therefore making the pollen break. The Ribolyser is made up from many little balls of ceramics and is used to homogenize biological samples. It works easily: you put a sample into the ribolyser and shake it well, best with a mechanical shaker. By shaking it, the little ceramic balls fly around in the container and by crashing into the sample, a lot of damage can be done to your biological sample. A negative side effect of using the ribolyser is that a lot of heat is produced when the balls fly around. We used the ribolyser to break the pollens wall with both heat and the mechanical damage done. Another way we tried to break open the wall of a pollen was by using liquid nitrogen. When you put liquid nitrogen onto cells, they break because all liquids touching that extremely cold substance freeze and eventually break. We helped that process by using a mortar.<br/> <br/>
  
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<b> <a href="https://2018.igem.org/Team:Rheda_Bielefeld/PCR" style="color:yellow;font:size:25px;"> PCR </a> </b> <br/> <br/>
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<b> <a href="https://2018.igem.org/Team:Rheda_Bielefeld/PCR" style="color:yellow;font-size:25px;"> PCR </a> </b> <br/> <br/>
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When we extracted the DNA from the leaves, we used a mortar, water, and some ethanol to gap the cellular wall of them. After crushing them we used filters from the "Nucleospin DNA Extraction Kit" to extract the DNA from the remains. When using that kit, you need two filters, one container for the flow-through, liquids included in the kit and a centrifuge. The kit basically binds the DNA to the filter, washes out other things in the filter and in the end, you use an elution buffer to elute the DNA from the filter. For the PCR, you have to prepare your sample by adding the nucleotides, the primer, the polymerase, and some water. Afterward, you put the samples into the PCR machine. It goes through many cycles of changing temperatures. In that process, the DNA parts into two strings and the primer binds onto their triplet and "shows" the polymerase where it has to set on. Next, the polymerase goes along the string and copies the sequences with opposing nucleotides. From that process, the DNA was multiplied. When the PCR has finished, we performed a gel electrophoresis. The gel electrophoresis consists of an agarose gel, TAE-buffer, an anode and a cathode, and a connection to electricity. Since the DNA has a positive charge, it is being moved towards the anode. The gel electrophoresis can be evaluated by putting it into a bath of a coloring chemical. The gel is afterward put into a dark chamber and gets lit by UV-Light. From that, the gel electrophoresis pictures resolve. <br/> <br/>
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<b> <a href="https://2018.igem.org/Team:Rheda_Bielefeld/Assays" style="color:yellow;font-size:25px;"> Assays </a> </b> <br/> <br/>
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<b> <a href="https://2018.igem.org/Team:Rheda_Bielefeld/Interlab" style="color:yellow;font-size:25px;"> InterLab Study </a> </b> <br/> <br/>
 
</article>
 
</article>
 
</div>
 
</div>

Revision as of 09:05, 17 October 2018

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Title

Text

Methods

We have used various different methods for our experiments. Here is an overview of the methods we have used.

Pollen

For our experiments with Pollens, we have used two methods. The first method was using trypsin and a ribolyser. Trypsin is an enzyme mostly found in the digestive system of many animals and can hydrolyze proteins. We have used it here in the hope that the trypsin would hydrolyze the proteins in the pollen´s outer wall and therefore making the pollen break. The Ribolyser is made up from many little balls of ceramics and is used to homogenize biological samples. It works easily: you put a sample into the ribolyser and shake it well, best with a mechanical shaker. By shaking it, the little ceramic balls fly around in the container and by crashing into the sample, a lot of damage can be done to your biological sample. A negative side effect of using the ribolyser is that a lot of heat is produced when the balls fly around. We used the ribolyser to break the pollens wall with both heat and the mechanical damage done. Another way we tried to break open the wall of a pollen was by using liquid nitrogen. When you put liquid nitrogen onto cells, they break because all liquids touching that extremely cold substance freeze and eventually break. We helped that process by using a mortar.

PCR

When we extracted the DNA from the leaves, we used a mortar, water, and some ethanol to gap the cellular wall of them. After crushing them we used filters from the "Nucleospin DNA Extraction Kit" to extract the DNA from the remains. When using that kit, you need two filters, one container for the flow-through, liquids included in the kit and a centrifuge. The kit basically binds the DNA to the filter, washes out other things in the filter and in the end, you use an elution buffer to elute the DNA from the filter. For the PCR, you have to prepare your sample by adding the nucleotides, the primer, the polymerase, and some water. Afterward, you put the samples into the PCR machine. It goes through many cycles of changing temperatures. In that process, the DNA parts into two strings and the primer binds onto their triplet and "shows" the polymerase where it has to set on. Next, the polymerase goes along the string and copies the sequences with opposing nucleotides. From that process, the DNA was multiplied. When the PCR has finished, we performed a gel electrophoresis. The gel electrophoresis consists of an agarose gel, TAE-buffer, an anode and a cathode, and a connection to electricity. Since the DNA has a positive charge, it is being moved towards the anode. The gel electrophoresis can be evaluated by putting it into a bath of a coloring chemical. The gel is afterward put into a dark chamber and gets lit by UV-Light. From that, the gel electrophoresis pictures resolve.

Assays



InterLab Study

Title

Text