Reducing costs and shortening the cetyltrimethylammonium bromide (CTAB) method to improve DNA extraction efficiency from wintersweet and some other plants

DNA extraction is a fundamental technique in molecular biology. For Chimonanthus praecox-a winter-flowering tree species-extensive and rapid DNA extraction is necessary to support genetic analyses. Currently, DNA extraction in C. praecox primarily relies on the traditional cetyltrimethylammonium bromide (CTAB) method, which is time-consuming and labor-intensive, hindering large-scale DNA extraction work. In this study, the different steps in the CTAB method are compared and evaluated to optimize the C. praecox leaf DNA extraction process. The water bath duration significantly impacts DNA extraction efficiency; the longer the water bath, the higher the DNA concentration. However, a 10-min water bath is sufficient to yield DNA of ideal concentration and purity. Additionally, a single extraction step is appropriate, with a 10-min precipitation at - 20 °C yielding high-quality DNA. Additionally, the pre-treatment step was modified by using a frozen pipette tip to crush samples directly in a centrifuge tube, reducing operational complexity and minimizing liquid nitrogen and sample consumption. Only 25 mg of sample is required, and high-quality DNA from C. praecox leaves can be extracted within 1 h. The amounts of required sample and liquid nitrogen were reduced by 75% and 90%, respectively. Moreover, the time required for the simplified extraction step was reduced by 77.14%. The applicability of the simplified scheme was evaluated using different C. praecox tissues, Calycanthaceae family members, and species of other families. The simplified scheme extracted DNA from the tepals and leaves of C. praecox with higher concentration purity. However, this protocol was biased toward the Chimonanthus family, Nicotiana tabacum, Populus tomentosa, and Lilium brownii. The proposed method enables the rapid and efficient extraction of high-quality DNA from 25 mg of plant leaves and is suitable for multiple species. This method reduces sample and liquid nitrogen consumption, lowering costs while significantly shortening the procedure time and enhancing extraction efficiency. This method is highly suitable for applications involving the extraction of large amounts of low-concentration DNA across various plant species.