Low DNA Yield from HMW Extraction (HMW DNA Extraction (Monarch))

Question

Accepted Answer

Recovered DNA concentration is significantly lower than expected based on input amount. Spectrophotometric measurements may show unexpectedly low concentrations for UHMW DNA samples. Common causes: 1) Input amount below recommended range (< 1 × 10⁵ cells minimum) 2) Lysis volume too large for the input amount, causing DNA concentration too low for efficient bead binding 3) DNA did not attach to beads and remains in solution during binding step 4) Incomplete binding to beads when working with high input (≥ 5 × 10⁶ cells or ≥ 1 ml blood) at low agitation speeds 5) UHMW DNA unevenly dispersed in solution, leading to inaccurate spectrophotometric quantitation 6) For blood: excess supernatant (> 20 µl) left after erythrocyte lysis inhibits DNA binding to beads Solutions: 1) Use recommended input amounts: 1–5 × 10⁵ cells (low input protocol), 5 × 10⁵–1 × 10⁷ cells (standard), or 100–2000 µl blood 2) Use appropriate lysis volume for input amount; reduce volume 3X for ≤ 5 × 10⁵ cells or < 500 µl blood 3) If DNA doesn't attach: twist tube sideways to create contact, or spin down precipitate, remove supernatant, wash with 500 µl gDNA Wash Buffer, air dry 5 min, elute at 56°C for 5–15 min with 300 rpm agitation 4) Increase binding time to 8 minutes in rotator for high input samples 5) For UHMW quantitation: take repeated measurements or shear small aliquot to homogenize before measuring 6) Remove erythrocyte supernatant carefully, leaving < 20 µl behind to minimize hemoglobin carryover