DNA extraction from green micro algae

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Amir_yam
Amir_yam's picture
DNA extraction from green micro algae

Hi everybody

I need to extract a genomic DNA from green micro algae.

I will be verey happy if anyone who have or can reach for me a protocol for this extraction.

thanks
Amir

Dilip Fernando
Dilip Fernando's picture
Dear Amir

Dear Amir
there are basic two methods for DNA extraction
Conventional method - this uses digestion buffer with proteinase k
Use of Kits - now several companies produces DNA extraction kits for instance DNAZole so You can buy them
If you need further info about Conventional method contact
this is the cheapest method

Wish you good luck
Dilip

Tony Rook
Tony Rook's picture
Amir_yam:

Amir_yam:

Here are some links to some publications regarding DNA extraction from algae:

Naomi Phillips, Celia M. Smith, Clifford W. Morden. An effective DNA extraction protocol for brown algae. Phycological Research 2001, 49 (2), 97102.
doi:10.1046/j.1440-1835.2001.00229.x

Abstract:
Successful extraction of total DNA from brown algae, which are generally polysaccharide and polyphenol rich, is often problematic using current methods. Persistent polysaccharide and polyphenolic compounds can hinder further application of modern molecular techniques requisite to molecular-based evolutionary studies. Our broad and long-term research goals with fucalean taxa, especially Sargassum, and problems with existing DNA extraction methods were an impetus to develop a reliable DNA extraction method. Initial research established hexadecyltrimethylammonium bromide (CTAB) based total-DNA methods as the most viable for further empirical development. Several constituents effective at either complexing secondary compounds or creating a reductive extraction environment were increased in concentration or added to the extraction buffer. These seemingly minor changes resulted in the creation of a highly reductive extraction buffer and effective total-DNA harvesting technique. We detail these modifications and demonstrate the reliability of the modified protocol with a variety of brown algae and tissue preservation methods. Such DNA is shown to be suitable for a variety of molecular techniques.

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Elena Varela-lvarez, Nikos Andreakis, Asunción Lago-Lestón, Gareth A Pearson, Ester A Serro, Gabriele Procaccini, Carlos M Duarte, Nuria Marbá (2006). GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION. Journal of Phycology 42 (3), 741745.
doi:10.1111/j.1529-8817.2006.00218.x

Abstract:
A method for isolating high-quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 610 μg genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A260/A280 ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum. The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.

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Gaoge Wang, Yuhui Li, Peng Xia, and Delin Duan. A simple method for DNA extraction from sporophyte in the brown alga Laminaria japonica. Journal of Applied Phycology. 2005, 17(1): 75-79.
DOI: 10.1007/s10811-005-5557-9

Abstract:
A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol (24:1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 μg g−1 (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A260/A280, which was about 1.71.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.

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Amir_yam
Amir_yam's picture
Thanks all for yours help.

Thanks all for yours help.

Amir

Tony Rook
Tony Rook's picture
And here are couple of links

And here are couple of links to online protocols and a link to a commercial kit capable of algae DNA extraction...

DNA EXTRACTION FROM MARINE ALGAE AND SEAGRASSES
Clark University - Worcester, MA
Deborah L. Robertson

Summary:
There are several published extraction methods that work well with marine algae and which have influenced the method described below (Coyer et al. 1994; Doyle and Doyle, 1987; Fain et al. 1988; Goff and Coleman, 1988; Herrin and Worley, 1990; Murray and Thompson, 1980; Polne-Fuller, 1991). This extraction protocol has yielded large quantities of DNA from a variety of algal species. For some species, the DNA can be used following ethanol precipitation but others require further purification of CsCl gradients for reproducibly high quality DNA.

DNA extraction from marine algae and seagrass is hampered by the large quantity of polysaccharides and polyphenolics produced within the thalli (leaves) of many species. The method uses the detergent CTAB and high salt concentrations to precipitate polysaccharides. CTAB forms complexes with polysaccharides at salt concentrations above 0.5 M at room temperature (Murray and Thompson, 1980). At lower salt concentrations, CTAB complexes with nucleic acids. PVPP and beta-mercaptoethanol are added to bind and precipitate polyphenolics.

Many steps in this protocol should be optimized for different algae depending on the characteristics of the thallus and polyphenolic content, but hopefully this outline will provide a starting point. The protocol assumes that the users are familiar with the general molecular procedures including the handling and disposal of chemicals.

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Host free - algal DNA extraction
Oregon State University - Virginia Weis Lab

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SoilMaster DNA Extraction Kit - Epicentre Biotechnologies Cat. Nos. SM02050, SM02005, SC04350 and SR04350

Summary:
The SoilMaster DNA Extraction Kit provides all of the reagents necessary to recover PCRready DNA from a variety of environmental samples. The kit utilizes a hot detergent lysis process combined with a chromatography step, which removes enzymatic inhibitors known to coextract with DNA from soil and sediment samples. The extracted DNA is PCR-ready, and can be used with the FailSafe PCR System to amplify bacterial, plant or fungal templates.

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Please let the rest of us on the board know which method worked the best for your application. This will greatly help anyone who may have this issue in the future.

Good luck!

Kuchi
Kuchi's picture
Hello i would like to isolate

Hello i would like to isolate genomic DNA from the green algal cultures that i have. Can u plz send me protocols for isolation & also guide me in purifying my cultures ( bacteria / fungal free )

surviving
surviving's picture
hi mr. tony rook,

hi mr. tony rook,

i really appreciate journals u have posted and find it very useful for me, unfortunately my institution do noy have subscription on the journal and email adds of the authors seems not working...could you lend me your personal copies of a) an effective dna extraction protocol for brown alage and b)genomic dna isolation from green alage and brown algae.

thanks