Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata
We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.
Key wordsGracilaria tenuistipitata plastid isolation plastid DNA purification ptDNA red alga Rhodophyta
Unable to display preview. Download preview PDF.
- Fain SR, Druehl LD and Baillie DL (1988) Repeat and single copy sequences are differentially conserved in the evolution of kelp chloroplast DNA. J Phycol 24: 292–302.Google Scholar
- Goff LJ and Coleman AW (1988) The use of plastid DNA restriction endonuclease patterns in delineating red algal species and populations. J Phycol 24: 357–368.Google Scholar
- Herrmann RG (1982) The preparation of circular DNA from plastids. In: Edelman M, Hallick R, and Chua NH (eds), Methods in Chloroplast Molecular Biology, pp 259–280, Elsevier, Amsterdam.Google Scholar
- Oliveira EC, Paula EJ, Plastino EM, and Petti R (1995) Metodologías para el cultivo no axénico de macroalgas marinas in vitro. In: Alveal K, Ferrario ME, Oliveira EC and Sar E (eds), Manual de Métodos Ficológicos, pp 429–447. Universidad de Concepción, Concepción, Chile.Google Scholar