Hi everyone,

I'm working on an in-field metagenomic DNA extraction protocol for ONT sequencing, at the moment on chicken fecal samples. I use a bead-beating + silica column based method, followed by AMPure XP cleanup to purify my DNA.

However, I'm never able to achieve A260/230 higher than 1.7, even after multiple rounds of AMPure XP cleanup.

The extraction method uses bead beating for sample homogenisation and lysis, followed by proteinase K digestion at RT. The digested sample is then slowly run over a silica column to purify the DNA, followed by some 70% ethanol washing steps. Despite the washing, the eluted DNA is highly impure (A260/280 1.7 and A260/230 1.0).

To increase purity, I perform multiple rounds of AMPure XP cleanup, including multiple 80% ethanol washing steps. This increases the A260/280 to 1.8-1.9, which is sufficient. However, the A260/230 tops of at 1.6-1.7. When sequencing my best samples using the ONT rapid sequencing kit, I observe rapid decline of pore activity (in only 12h, 0/1300 active pores were left).

What could be the contaminant that causes this low A260/230 after the AMPure cleanup?

Are there any suggestions to remove this contamination, preferably not requiring centrifugation at >8000g (for in-field applicability)?

Thank you!