Large-scale extraction of pure DNA from mature leaves of Cyclamen persicum Mill. and other recalcitrant plants with alkaline polyvinylpolypyrrolidone (PVPP)

We present a set of DNA extraction/purification/quantification procedures for preparation of largeamount and pure DNA samples from mature leaves of cyclamen and tree species, from which high quality and quantity of DNA would not be extractable with any other protocols. Cyclamen is a popular flowering plant throughout the temperate world. Only minute amounts or non-detectable levels of cyclamen DNA can be extracted from its leaves by standard methods such as the CTAB method. We have developed a new DNA extraction buffer which we call the 'PVPP buffer'. This buffer can extract 20 lig of DNA from 1 g fresh weight of mature cyclamen leaves. Following extraction, DNA was successfully purified by CsCl ultracentrifugation, but purification was not successful with CTAB precipitation. Qualitative and quantitative analyses of the DNA extract were performed by gel electrophoresis, not by the popular UV absorbance-based analysis, which seems to overestimate DNA quality and quantity. This fact suggests the possibility that quality and quantity of DNA extracts were not necessarily enough in the former reports. Pure DNA in large amounts was also extractable from mature leaves of all species testea (camellia, chrysanthemum, orchid, pine, rose and tea), which include 'recalcitrant' ones, using the PVPP buffer and CsCI purification. Extraction using PVPP buffer does not require special equipment and CsCI purification requires access to an ultracentrifuge. (C) 2013 Elsevier B.V. All rights reserved.