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project:biolab:start [2025/03/22 15:40] – [Platyhelmithes / Flatworms] sumie-dhproject:biolab:start [2025/06/15 07:54] (current) – sachy
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 ===== BioOSM ===== ===== BioOSM =====
  
-Some members are collecting photos and other evidence of (micro)organisms which can (not) be seen from behind the computer screen. Please see the [[project:bioosm:start|BioOSM project pages]].+Some members are collecting photos and other evidence of (micro)organisms which can (not) be seen from behind the computer screen. Please see the [[project:bioosm:start|BioOSM project pages]]. Project is closely knitted with Planarian working group.
  
 ===== Planarian working group ===== ===== Planarian working group =====
  
-External project led by sumie-dh. Project is  beyond 'normal' naturalist way of doing and following certain goals and plans. If you want taste topics like how to deal with unknown [possibly] new species in your collections, how to manage fieldwork plans, messing with keys and searching for resources, research ideas and workflow designs in taxonomy, curating of own sample collection in +- standardized ways, WSI processing and dealing with presentation of your results outside of 'hackerspace setup' talk to members of planarian working group > [[user:sumie-dh]]| [[user:sachy]]. +External project led by sumie-dh. Project is  beyond 'normal' naturalist way of doing and following certain goals and plans. If you want taste topics like how to deal with unknown [possibly] new species in your collections, how to manage fieldwork plans, messing with keys and searching for resources, research ideas and workflow designs in taxonomy, curating of own sample collection in +- standardized ways, WSI processing and dealing with presentation of your results outside of 'hackerspace setup' talk to members of planarian working group > [[user:sumie-dh]]| [[user:sachy]]. Forget anything DIY in this project ;] 
  
 Overview of currently solved topics is on: https://bioosm.brmlab.cz/histolka/project_imagery/project_imagery.htm Overview of currently solved topics is on: https://bioosm.brmlab.cz/histolka/project_imagery/project_imagery.htm
-Note this is only fragment of running things, we going on own pace, slowly but surely forward, this is speed of planarian.  +Note this is only fragment of running things, we are going on own pace, slowly but surely forward, this is speed of planarian.  
  
 +Onboarding of new members: unlikely but discuss it with members. 
 +
 +Planarian samples welcomed but keep permit logic in mind in case of foreign samples or those from local national parks.
 + 
 Finished fieldwork sessions: Finished fieldwork sessions:
  
-2024: First record of the land planarian species Rhynchodemus sylvaticus (Leidy,1852) in a naturalized area of Czechia  [manuscript soon, waiting for DNA barcoding] +  * 2024: First record of the land planarian species Rhynchodemus sylvaticus (Leidy,1852) in a naturalized area of Czechia  [manuscript soon, waiting for DNA barcoding] 
-2023-2024: Sexual strains of Girardia tigrina in Central Bohemian region - self-funded, further work ongoing +  * 2023-2024: Sexual strains of Girardia tigrina in Central Bohemian region - self-funded, further work ongoing 
-2022: Mapovánı́ ploštěnky Dugesia gonocephala v povodı́ Svinenského potoka - grant CSOP_122210, further work ongoing +  * 2022: Mapovánı́ ploštěnky Dugesia gonocephala v povodı́ Svinenského potoka - grant CSOP_122210, further work ongoing 
-2021: Mapovánı́ ploštěnky Crenobia alpina v okolı́ Hojné vody - grant CSOP_122123+  * 2021: Mapovánı́ ploštěnky Crenobia alpina v okolı́ Hojné vody - grant CSOP_122123
  
 ===== Epoxidation of samples ===== ===== Epoxidation of samples =====
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 ===== PCR and electrophoresis ===== ===== PCR and electrophoresis =====
  
-DNA electrophoresis using the OpenPCR [[http://openpcr.org/]] +See [[.pcr]]
- +
-=== Status === +
-  * openPCR working, dry run successful (SW: [[https://github.com/cathalgarvey/OpenPyCR]], running.) +
-  * Taq/dNTP PCR mix obtained from [[http://www.openbiotech.com]] +
-  * electrophoresis working, tested at hackathon +
-  * got chemicals: +
-    * TAE buffer [[http://letters.cathalgarvey.me/cargo-cults-and-electrophoresis/]]) +
-    * ddH2O +
-    * PCR grade agarose (quite little, expensive) +
-    * EtBr +
-    * SYBR Gold (thanks [[user:Mrkva]] and [[user:barney]]!) +
-    * some DNA fragments for experimentation (pTracer plasmids) +
-  * freezer (-20°C) for DNA/enzyme storage is ready +
-  * we have a wonderful power supply (up to 300V) thanks to Tomsuch +
-  * transilluminator built from blue LEDs  +
- +
-the first test run with crude agar, self made TAE, DIY loading dye (cresol red+sucrose), 60V (with the power source from hwlab), 1.5h: +
- +
-{{:project:biolab:img_6511.jpg?200|}} +
- +
- +
-{{:project:biolab:img_6512.jpg?200|}} +
- +
- +
-second test run with agarose and several restriction digest samples using a strong laser and SYBR gold for visualization - blue light filter from infra-soldering station: +
- +
-{{:project:2802_e4dd_960.jpeg?nolink&300 |}} +
- +
- +
-some notes for using SYBR gold:  +
-  * DO NOT STORE IN GLASS CONTAINER +
-  * protocol: [[https://tools.lifetechnologies.com/content/sfs/manuals/mp11494.pdf]]  +
- +
-**first test run of the openPCR**   +
- +
-Template: pTracer plasmid +
- +
-Primers: inf pTracer F + R (10uM) designed by [[user:chido]] to amplify the GFP/Bsd gene  +
- +
-PCR mix: +
-  dH2O 33ul +
-  mastermix Openbiotech 5x 10 ul +
-  primer F 2ul +
-  primer R 2ul +
-  template 1ng/ul 3ul +
- total volume 50 ul +
- +
-Thermocycler settings: +
-  95 °C 60 sec +
-    95 °C 30 sec +
-    60 °C 30 sec +
-    72 °C 60 sec +
-  x30 +
-  4 °C 20 sec  +
- +
-SYBR gold gel stain: +
-  30 ml TAE, 0.3g agarose gel +
-  stained in 10ul SYBR gold diluted in 100ml TAE, 40 min  +
- +
-Results:  +
- +
-We obtained the desired product using Openbiotech master mix that has been stored in -20 °C, but there was no product at all when using a mastermix that has been stored in the fridge. We aliquoted both SYBR gold gel stain and the PCR mastermix so thawing the stock solution is not necessary.  +
-OpenPCR works fine! Pictures and analyzed gel coming soon. Next step: devise a protocol for DNA isolation that can be used as a PCR template. +
- +
-Useful links: +
-  * primer design guide: [[http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html]] +
-  * paper on horses/rats/cockroaches detection in food via PCR [[http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf]]    +
-  * there are online companies that will synthetize custom primers for a fee [[http://www.operon.com]][[http://www.biogen.cz]]+
  
 ===== Fun with Bioluminescence ===== ===== Fun with Bioluminescence =====
project/biolab/start.1742658038.txt.gz · Last modified: 2025/03/22 15:40 by sumie-dh