Optimizing the lysis step in CTAB DNA extractions of silica-dried and herbarium leaf tissues

Abstract

Premise: The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality. Methods and Results: We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel–preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium-preserved tissues. Conclusions: We recommend DNA extractions of silica gel–preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time.