LAB # 5 MARINE BIOTECHNOLOGY

1. LAB # 5MARINE BIOTECHNOLOGY MACROALGAE

2. Introduction • Algae: a large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms. • Macroalgae(Seaweeds): a macroscopic, multicellular, benthic marine algae.

3. Red algae (Rhodophyta) • Brown algae (Phaeophyta)

4. Green algae (Chlorophyta)

5. Macro algae uses: • Food: Nori (sushi)(red algae) • Agar production: Gelidium sp. • Medicine: production of dental moulds & cosmetics

6. Fertilizer: treatment of waste water to make it suitable for human use • Energy source: Algae fuel, source of bioethanol (gasoline) • Pollution control: remove nutrients & other pollutants from wastewaters.

7. Today’s lab Objective: • DNA extraction from the green algae Enteromorpha sp. by using CTAB extraction buffer. • CTAB is a useful detergent for isolation of DNA from tissues containing high amounts of polysaccharides. • The presence of polysaccharides in a DNA preparation can inhibit use of techniques such PCR. • Under the high-salt conditions used in this protocol, the CTAB binds the polysaccharides, removing them from the solution.

8. Today’s sample (Enteromorpha flexuosa) • A common green algae found wherever there is freshwater stream or underwater spring input to the ocean. It is often associated with coastal areas of high nutrients, including areas with residential and industrial development.

9. Buffers: 2x CTAB extraction buffer • 100 mM Tris-HC1 (pH 8.0) • 1.4 M NaC1 • 20 mM EDTA • 2% CTAB • 0.2% B-mercaptoethanol TE buffer • 10 mM Tris-HC1 • 1 mM EDTA • 1 M NaCI, pH 8.0

10. Sample preparation • Freeze pieces of algal tissue in liquid nitrogen, mixed with a small amount of dry ice, and ground to a fine powder using a mortar and pestle. • Transfer the ground tissue dry ice mixture quickly to sterilized tubes, & place it at -70 C with the tops open. • Cap tubes after sublimation of the dry ice, and stored at -70 C until needed for DNA extraction.

11. Algae DNA extraction protocol 1. Preheat CTAB extraction buffer in water bath at 65°C. 2. Green algae were ground by a tissue pestle with 1ml of CTAB extraction buffer. 3. Incubate the sample tube at 65°C for 30 min. 4. After incubation the mixture was cooled at RT. 5. Equal volume of the mixture of chloroform : isoamylalcohol (24 : 1) was added & mix by inversion for 10 times. 6. The mixture was centrifuged at 12000 rpm for 10 min at 25°C.

12. 6- Transfer the aqueous phase to a fresh tube & add 0.6 volumes of ice-cold isopropanol for DNA precipitation & store at –20°C for 30 min. 7. Centrifuge the precipitated DNA at 9000 rpm for 10 min at 4°C. 8. Transfer the supernatant carefully and wash the pellet with 70% ethanol, & dry the pellet at RT for 10 min. 9. Dissolve the pellet in 50 ul of 1x TE buffer & store samples at -20 C until use.

13. Further analysis: • After genomic DNA was isolated from fresh material, the quality and the size of DNA were checked by agarose gel against a known molecular marker. • Quantification of DNA concentration was performed by spectrophotometer measurement of UV absorbance