2025-03-21
This is Simon's COMMONS Lab daily Open Notebook.
Today is 2025.03.21
Todo today
Writing the methodology for the sampling As I don't know how to create new files in my bachelor work's dendron. I will start writing the methodology here and paste it in the dendron later. Of course, it will be heavily inspired by what Héloïse and Edouard already did
Have a look at the COMMONS research discussion forum
- https://github.com/orgs/commons-research/discussions
Doing
My methotodology for the tree extractions so far: My project will focus on the extraction of tree metaboloms and the continuation, and hopefully the end, of the DBGI project in Fribourg. Unlike,the majority of plants already sampled, trees are perennial and tend to present harder parts (bark, trunk, ...). This presents a first challenge for the already tested DBGI method. As of now, the method defines each sample as being its own unit. In the case of trees, this simplification is somewhat limiting as the different organs (which might be quite numerous) can't all be linked to a single individual through the already established informatical system. The hardness of the tissues could also cause a problem by not breaking properly with the metal beads. However, this issue might be overcome by the use of disc shaped beads that should slice right through.
All the (remaining) trees in the botanical garden need to be sampled for this bachelor's work. They are scattered all over the garden, with a greater concentration in the arboretum, and near the fences as hedges. They tend to come from all over the world with local species (Picea abies) aswell as tropical ones (Persea americana). How are they placed in the garden ? Taxonomic/usefulness reasons? Remains of the old organisation of the garden ?
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Sampling
The classical DBGI methodology was mostly applied to collect the different organs. The main difference being the use of ladders, and telescopic cutting tools. For each sample, the geographical position of the specimen will be marked down on a QField map. Additional data, such as photos: 1) the identification panel 2) the identification panel with the number of the sample 3) photo of the whole specimen 4) a detailled photo of the leaves/flowers/seeds/fruits, 5) the sampled zone 6) Additional photo, if judged necessary will be taken.
The photos, and the location will then be imported on iNaturalist for receving further confirmation of the species. This, in term, should allow the identification of sampled species in the wild without having to take an expert in the field A branch will be cutted down. From the fallen branch, leaves/needles, bark, twigs, flowers, fruits will be collected. => this will need to be rewritten after the first samplings. After the cut, the organ is wrapped in a brown coffee filter, and shoved in a falcon tube closed with a perforated cap. Each tube is pre-labeled with a unique QR-code that will allow to identify and track the sample through the extraction process and the storage. The tube are then dunked in liquid nitrogen. They will finally be put to rest in the freezer (-80C), if they can't be freeze-dryed immediately. The freeze-drying process needs to be at least 72 hours long to make sure the samples is absolutely dry. Directly after the freeze-drying process, all the perforated caps need to be switched out with standard, desinfected, non-perforated caps. The falcon tubes are then stored in a labeled rack. They need to be scanned to allow the system to track them.
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Extraction
The first step of the extraction process is weighing out 50 milligrams (!!0.0500 g sur la machine!!) of each sample. An error of 5% (+-2,5mg) was accepted. The matter is weigthed in a 2 ml Eppendorf with a rounded bottom. Depending on the weight, metal beads are added. Less than 20mg =1; between 20mg-50mg = 2beads, around 50mg= 3 beads(this may change with the new discoid beads). => shape of beads depends on type of sample.
The samples go in the MM400 Retsch machine for 2.5 minutes at 25Hz. Then, 1,5ml(why not the same as Héloïse and Edouard's protocol ?) of the classic DBGI extraction solution. It is a mix of 80% methanol, 20% distilled water, and 0,1% formic acid. The newly rehydrated samples go back in the Retsch machine with the same settings. The tubes are then taking a turn in a centrifugation machine for 2 minutes at 13'000 RPM to separate the supernatant from the plant deposit. Finally, as much of the supernatant (usually 1,4 ml) is collected and pipetted in a glass vial with a hermetically sealed cap (the red ones). The vial need to be labelled and associated to a container ensuring the tracking of the sample from the garden to the MS. This final step needs to be performed under a bench. The vials container is then stored in the freezer (-80C), and is ready for MS and further analysis.
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Analysis ?
Paused
Done
Notes
Todo tomorrow, one day ... or never
[] Start the collect of differents tree organs (bark if possible, leaves, seeds, flowers/reproductive organs, buds, twigs/small branches) []Continue with sample preparation [] Download Zotero ? [] Check the list of plants, and trees of the garden [] Find a way to automatically copy/paste what is in the to do list of the previous entry into the newest log [] When grinding woody parts for the first time, check if they need to rest a longer time in the extraction solution (cells not well enough broken). Or if the extraction process can follow up directly. [] Check the list of trees on the BGUNIFR Botalista [] Need to book the freeze-dryer for friday 28 [] Notify Edouard that I might have fucked up on the units announced in the vial app