Hello everyone,

I typically obtain good results using the commercial silica column-based DNA extraction kit from QIAGEN (dried mushroom specimens and pure cultures). However, I’ve recently encountered unusually poor yields and purity values.

In several recent extractions, DNA concentrations were below 20 ng/μl (some as low as 0.6–10 ng/μl), with A260/A280 ratios between 1.3 and 1.5 (measured with a Nanodrop Lite). This is surprising, as the DNeasy kit is usually quite reliable, even for difficult samples, which aligns with its high cost.

My protocol includes:

• Grinding samples with liquid nitrogen until a fine powder
• Incubation with extraction buffer + RNase at 65 °C for ~2.5 hours
• A third centrifugation after the AW2 wash step to remove residual ethanol
• Two elution steps with 20 μl of pre-warmed AE buffer (40 μl total), with a 10-minute incubation before each spin

What’s puzzling is that even recently subcultured fungal isolates are producing very low yields and poor purity ratios.

Could this be due to using too much starting material? Are there other common causes for this sudden drop in extraction quality (e.g., kit degradation, changes in sample composition, etc.)?

I’d appreciate any thoughts or similar experiences. Thanks in advance!