Preparation of high molecular weight genomic DNA from nuclei of woody plants

Abstract

We have developed a rapid and reliable method for preparation of high molecular weight genomic DNA from sweet orange (Citrus sinensis) suitable for subsequent digestion by rarely cutting restriction enzymes and then separation by pulsed-field gel electrophoresis (PFGE). Methods previously described for preparation of plant DNA prior to PFGE involved protoplast isolation, a procedure that can be inefficient and time-consuming for several plant species. Nuclei isolated from plant tissues were embedded into agarose blocks and treated to release DNA, which was cleaved by restriction enzymes and then submitted to PFGE. One gram of fresh leaves gave approximately 15 micrograms of high molecular weight genomic DNA (> 2000 kbp). Within-gel hybridizations were used instead of classical Southern blotting, and the resulting signals were adequate when they were compared with those obtained with DNA prepared from crude ground leaf tissues.