Most extraction failures are not random. They follow predictable patterns. These are the five we see most often, and what drives each one:

1. Too much sample.

More starting material does not always mean more usable DNA. Overloaded extracts carry inhibitors into the reaction at concentrations that suppress PCR. For enzymatic methods, the fix is almost always dilution, typically 1:5, rather than a protocol change. Test by spiking the extract with a known positive control: if PCR recovers, the extract is inhibited not absent.

2. Chelating agents stripping enzyme cofactors.

EDTA is a common blood anticoagulant and storage reagent. It chelates calcium, which the thermophilic proteinase requires for activity. If using EDTA-collected blood or buffers containing EDTA, add 0.02 volumes of 10 mM CaCl2 before extraction. CaCl2 is included in blood extraction kits for this reason.

3. Inhibitors from sample material.

Cigarette tar, plant polyphenols, bone matrix compounds, and humic acids in soil all inhibit PCR. BSA (1 µl of 10 mg/ml per 25 µl reaction) reduces inhibition in the amplification step without modifying the extraction. Dilution helps further. The issue is downstream, not extraction failure.

4. Incomplete cell wall disruption in difficult substrates.

Gram-positive bacteria, fungal material, and plant tissue all require additional enzymatic preparation before the thermophilic proteinase can access genomic DNA. Using 𝘱𝘳𝘦𝘱GEM Bacteria or 𝘱𝘩𝘺𝘵𝘰GEM which include mesophilic cell wall degrading enzymes at lower temperatures, addresses this directly.

5. DNA degradation before extraction begins.

Heat, nucleases, and desiccation all degrade DNA before the extraction kit is opened. For field-collected material, storage on FTA or silica cards, immediate freezing, or use of a stabilisation buffer matters as much as the extraction method itself.