DNA Fragmentation and Size Reduction

Symptom

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.

1. 1 Extended heating of HMW samples: > 15–30 min at 56°C or > 1–3 hours at 37°C
2. 2 UHMW DNA pipetted without wide-bore tips or vortexed, causing shearing
3. 3 Blood samples thawed before adding RBC Lysis Buffer, activating nucleases
4. 4 Fresh blood older than one week showing progressive degradation
5. 5 Tissue samples not homogenized immediately or not placed in thermal mixer immediately after homogenization
6. 6 Frozen tissue samples thawed before processing, allowing nuclease access to DNA

1. 1 Limit incubation times: maximum 15–30 min at 56°C, 1–3 hours at 37°C, or overnight at room temperature only
2. 2 Always use wide-bore pipette tips for UHMW DNA; avoid vortexing
3. 3 For frozen blood: keep frozen, add cold RBC Lysis Buffer directly to frozen sample
4. 4 Process fresh blood within one week of collection
5. 5 Place tissue samples in thermal mixer immediately after homogenization to inactivate nucleases rapidly
6. 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