Hi, I’m working with peripheral blood samples from patients with nephrotic syndrome to extract genomic DNA. I’m using a commercial kit based on cell lysis, proteinase K digestion, and ethanol precipitation. The samples have been processed under seemingly identical conditions (same reagents, times, temperatures, and volumes), but some show clear degradation of DNA on a 1% agarose gel, while others do not (for example, sample 18, which was re-extracted under the same conditions, appears intact).

I’ve considered several variables, but I would appreciate more input. Here's what I’ve observed:

• DNA was stored at –20 °C after elution.
• Nuclease-free water was used for elution.
• Elution was done at 65 °C for one hour, 300 RPM.
• GelRed was applied shortly before loading to avoid evaporation.
• Despite degradation on gel, spectrophotometric measurements (A260/A280 and A260/A230) were within acceptable purity ranges.

What technical or biological factors could be causing this selective DNA degradation?

I would greatly appreciate any insights on critical steps where DNA integrity might be compromised, even if reagents and protocol steps are consistent.

Thanks in advance for your help.