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

Revision as of 08:33, 17 October 2018

Extracting Pollen

Firstly, we gave the male syncarps of birch, willow and spruce in a plastic tube, which was electrostatically loaded on its walls. During vortexing the pollen stuck to the walls and the syncarps were collected at the bottom of the container. Afterwards, the pollen were wiped from the walls with cotton buds and the cotton pellets with the pollen were vortexed. Lastly, we checked the pollen using light-microscopy if they were separated correctly according to their plant species.

Opening Pollen

In order to isolate and to be able to analyse the DNA we tried to open the pollen in different ways. We used ribolyser tubes, small ball mills used to homogenize biological samples. We also added Trypsin to pollen in order to decompose parts of the exine (outer layer of pollen). Another idea was to liquid nitrogen, to cool down the cell walls and to break them open.
To form a trypsin solution, we resuspended 20µg of trypsin in 200µl of H20. We then split the solution into 4 eppis with 50µl each, having approximately 5µg of trypsin within every eppi. According to our proportionality-calculations we then added pollen to the trypsin and incubated at 37°C overnight. The following day we analysed our samples with light microscopy, enlarging by 100 and 400.
To use ribolyser tubes we added 10, 20 and 50 mg of pollen combined with 400 µl of water into the tubes. In the next step we shook the tubes in the ribolyser and centrifugated at 110 rpm for 10 minutes. Result were two different layers, one brighter than the other. On the top layer was white shroud. After abstracting the light-brown, lower, layer with a chemical dropper, 400µl lysebuffer were added and the solution was centrifugated for 1 minute with 11000 rpm. Afterwards, we used the machery nagel plant Nucleo spin III Kit for the DNA extraction.
Lastly, we tried to open the pollen with liquid nitrogen. Therefore, we put pollen in a small bowl and added liquid nitrogen. We then cracked the pollen using mortar and pestle and checked the results of all approaches with the light microscopy.

with nitrogen opened pollenof a spruce

Gallery

Here is the result from trying open the pollens with trypsin and a ribolyser.

pollen of a willow x400

pollen of a spruce x400

pollen of a birch x100

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 To use ribolyser tubes we added 10, 20 and 50 mg of pollen combined with 400 µl of water into the tubes. In the next step we shook the tubes in the ribolyser and centrifugated at 110 rpm for 10 minutes. Result were two different layers, one brighter than the other. On the top layer was white shroud. After abstracting the light-brown, lower, layer with a chemical dropper, 400µl lysebuffer were added and the solution was centrifugated for 1 minute with 11000 rpm. Afterwards, we used the machery nagel plant Nucleo spin III Kit for the DNA extraction.
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-Lastly, we tried to open the pollen with liquid nitrogen. Therefore, we put pollen in a small bowl and added liquid nitrogen. We then cracked the pollen using mortar and pestle and checked the results of all approaches with the light microscopy.
+Lastly, we tried to open the pollen with liquid nitrogen. Therefore, we put pollen in a small bowl and added liquid nitrogen. We then cracked the pollen using mortar and pestle and checked the results of all approaches with the light microscopy. <br><br><br><br>
+<img src="https://static.igem.org/mediawiki/2018/e/e6/T--Rheda_Bielefeld--StickstoffFichte400.jpg" style="width:400px;height:auto;"></img><br>with nitrogen opened pollenof a spruce
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