Abstract
Chlamydomonas reinhardtii is a soil-dwelling eukaryotic green alga that is widely used as a laboratory model organism for research on photosynthesis, ciliary biology, lipid metabolism and many other aspects of cell biology and physiology. With sequenced nuclear, chloroplast and mitochondrial genomes, Chlamydomonas is also an excellent organism for genetics and genomics research. This protocol describes the isolation of genomic DNA from Chlamydomonas using a standard phenol:chloroform extraction method followed by ethanol precipitation. The protocol requires minimal lab materials, takes approximately 4 h to complete, and can also be used for isolation of genomic DNA from other eukaryotic green algae.
Keywords: Algae, Genomic DNA, Phenol/chloroform extraction, Nucleic acid, DNA purification
Background
Isolating nucleic acid is a critical first step for cloning and sequencing genetic material and provides the basis for diverse molecular biological studies ranging from gene expression to gene evolution. A number of protocols exist for isolating DNA from algae (Weeks et al., 1986; Fawley and Fawley, 2004; HwangBo et al., 2010). Generally, cells are pelleted by centrifugation and lysed in buffer containing detergents such as SDS to solubilize membranes. This is followed by at least one extraction in phenol:chloroform and at least one extraction in chloroform. In some cases, an RNase treatment step is performed to degrade RNA. DNA is then precipitated by addition of ethanol or isopropanol and incubating on ice or in the freezer. After pelleting and washing the precipitated DNA, it is usually resuspended in water or buffer (e.g., Tris-EDTA) and quantified spectrophotometrically at 260 nm. The protocol described herein makes use of two phenol/chloroform/isoamyl alcohol extractions and two chloroform/isoamyl extractions, with an RNase treatment step in between. The addition of isoamyl alcohol to the organic solvents prevents foaming and stabilizes the interphase, which contains a high concentration of coagulated proteins. Importantly, this protocol results in high quality genomic DNA that is suitable for downstream applications such as cloning and sequencing.
Materials and Reagents
Equipment
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Acknowledgments
This work was supported in part by Howard Hughes Medical Institute funding awarded to Joseph P. Noel. Author declares no conflicts of interest or competing interests.
References
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