Request for Guidance: No DNA Yield After CTAB Extraction from Fungal Sample

[Mazhar Ali]

I hope this message finds you well. My name is Mazhar Ali, and I am currently working on fungal DNA extraction as part of my undergraduate research project. I recently followed a two-day CTAB-based protocol for extracting DNA from fresh fungal growth from petri plates (not mycelium). I used glass powder during grinding and included Proteinase K incubation overnight, followed by PCI extraction, ethanol washing, and gel electrophoresis.

However, I was unable to detect any DNA bands after running the gel. I would be sincerely grateful if you could help me identify potential mistakes or missing steps in my protocol.

Summary of My Procedure:

• Used fresh fungal colonies (not dry mycelium)
• Ground samples with glass powder in 300 µL CTAB buffer (65°C)
• Added Proteinase K and incubated at 56°C overnight
• PCI treatment and centrifugation (13,000 rpm, 10 min)
• Isopropanol precipitation and ethanol wash
• DNA resuspended in 50 µL nuclease-free water
• Loaded 3 µL sample on 1% agarose gel (with EtBr), but no bands appeared
  I would greatly appreciate your expert opinion and any suggestions for troubleshooting or improving the method. If needed, I can also share images of my gel or details of the reagents used.
  Thank you very much for your time and guidance.
  Best regards,

  Mazhar Ali

  BS Biological Sciences
  University of Veterinary and Animal Sciences, Lahore
  Email: mazhara...@gmail.com
  Phone: +92 3221404028