Room temperature lysis incubation improves DNA yield in the Norgen Plant/Fungi DNA Isolation Kit across multiple plant species

Abstract Premise Commercial plant DNA extraction kits typically include a 65°C lysis incubation step, inherited from cetyltrimethylammonium bromide (CTAB)‐based methods. Recent work suggests that cooler lysis temperatures can improve DNA purity in CTAB extractions; whether this also improves yield in CTAB‐based column kits has not been tested. Methods and Results We compared room temperature (RT, ~25°C) and 65°C lysis incubation using the Norgen Plant/Fungi DNA Isolation Kit (CTAB‐based lysis buffer) across six plant species in a paired split‐tissue design ( n = 59 pairs). RT incubation significantly increased Qubit fluorometric DNA yield by 47% overall, with a significant treatment × species interaction. RT‐extracted Cucurbita pepo DNA was compatible with genotyping‐by‐sequencing. In a focused generalization test, the modification did not improve yield with the Qiagen DNeasy Plant Mini Kit, whose lysis buffer uses the anionic detergent sodium dodecyl sulphate, indicating that the effect is specific to kit lysis chemistry. Conclusions For the Norgen kit, omitting the heating step is a zero‐cost modification that significantly improves DNA yield and the ratio of absorbance at 260 nm and 230 nm (A 260 /A 230 purity ratio) for most eudicot species tested, with no loss of downstream compatibility. This benefit appears specific to CTAB‐based lysis and should be validated for each kit and species before adoption.