DNA Fragmentation and Size Reduction (HMW DNA Extraction (Monarch))

Question

Accepted Answer

Extracted DNA shows reduced fragment size on gel electrophoresis or pulse-field gel analysis. UHMW DNA appears degraded with smaller molecular weight distribution than expected. Common causes: 1) Extended heating of HMW samples: > 15–30 min at 56°C or > 1–3 hours at 37°C 2) UHMW DNA pipetted without wide-bore tips or vortexed, causing shearing 3) Blood samples thawed before adding RBC Lysis Buffer, activating nucleases 4) Fresh blood older than one week showing progressive degradation 5) Tissue samples not homogenized immediately or not placed in thermal mixer immediately after homogenization 6) Frozen tissue samples thawed before processing, allowing nuclease access to DNA Solutions: 1) Limit incubation times: maximum 15–30 min at 56°C, 1–3 hours at 37°C, or overnight at room temperature only 2) Always use wide-bore pipette tips for UHMW DNA; avoid vortexing 3) For frozen blood: keep frozen, add cold RBC Lysis Buffer directly to frozen sample 4) Process fresh blood within one week of collection 5) Place tissue samples in thermal mixer immediately after homogenization to inactivate nucleases rapidly 6) Keep frozen tissue samples frozen; work with smallest tissue pieces for rapid Proteinase K inactivation of nucleases; snap freeze in liquid nitrogen to minimize ice crystal damage