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project:biolab:start [2025/03/22 16:25] – [Planarian working group] sumie-dhproject:biolab:start [2025/09/01 14:04] (current) – sachy
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 ~~META: ~~META:
-status = alive and kicking!+status = active
 &relation firstimage = :project:10076716473_fb5380cf36.jpg &relation firstimage = :project:10076716473_fb5380cf36.jpg
 ~~ ~~
 +---- dataentry project ---- 
 +name: Project Biolab 
 +status: active 
 +image: {{:project:dna.jpg}} 
 +----
  
  
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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.1742660743.txt.gz · Last modified: 2025/03/22 16:25 by sumie-dh