Rapid DNA Extraction from Plants

1 Introduction The first step of RAPD fingerprinting is the preparation of the target DNA template. Intuitively, minimal DNA template preparation should be necessary for RAPDs since, theoretically, PCR may amplify a single DNA molecule. It seems one would simply homogenize tissue and allow the PCR to "find" and amplify the target DNA. Indeed, many rapid DNA isolation methods designed for use with PCR actually involve little isolation of DNA. Rather, they employ a "grind and use" process (e.g., Wang et al. 1993) or minimal purification (e.g., Edwards et al. 1991). These methods are very fast, require little tissue and amplify well with plants that are amenable to DNA extraction and that contain few interfering secondary metabolites. However, the resulting DNA templates are not very pure and may not be stable for long periods of time. Recently, DNA purity has been implicated as one of the most important factors in RAPD reproducibility (McClel-land and Welsh 1994). McClelland and Welsh (1994) suggest that researchers use only high quality templates to assure reproducible RAPDs. The assay they suggest consists of replicate RAPD PCRs in which the DNA concentration is titrated over two orders of magnitude. If the DNA is of adequate quality the replicates should yield identical RAPD fingerprints. In addition, Heinze (1994) found that the addition of RNAse A improved RAPD reproducibility with gym-nosperm embryos. However, the basal method was a crude prep similar to that of Edwards et al. (1991). Thus, it seems-likely that there could be several interactive factors involving DNA purity or lack thereof in RAPD reproducibility. It seems prudent to researchers performing RAPD PCR to assure themselves that the isolated DNA is of sufficient quality to ensure reproducible RAPD fingerprints. The objective of this chapter is to describe a rapid CTAB miniprep that yields relatively pure DNA from plants and that can also be used