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project:biolab:start [2025/03/22 15:59] – [Planarian working group] 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 =====
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 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 are 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.+ 
 +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:
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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.1742659147.txt.gz · Last modified: 2025/03/22 15:59 by sumie-dh