This extraction method is based on previously published protocols (Doyle and Doyle, 1987) and works well for a variety of marine phytoplankton. The method yeilds DNAsuitable for many molecular applications including PCR and Southern blotting. The DNA can be further purified using the methods described by Coyer et al., 1994.

BUFFERS:

2x CTAB Buffer

100 mM Tris pH 8.0

1.4 M NaCl

20 mM EDTA

2% CTAB

0.1% PVPP

Add beta-mercaptoethanol to 0.2% (v/v) daily.

TE

10 mM Tris (pH 8.0)

0.1 mM EDTA

METHOD:

1. Add 10 mL of 2 x CTAB buffer per 1L of collected cells.
2. Incubate at 60 C for 0.5-1 h.
3. Add an equal volume of chloroform:isoamyl alcohol (24:1), mix very well.
4. Centrifuge for 10 min in 5,000 x g
5. Transfer aqueous phase (upper layer) to new tube. Repeat chloroform:isoamyl alcohol extraction (steps 3 and 4)until the interface is clean.
6. Add 2/3 volumes of isopropanol, mix gently.
7. Centrifuge for 15 min at 10,000 x g. Discard supernatant and wash pellet with 70% ethanol.
8. Air dry pellet. Resuspend in TE to appropriate concentration.
9. DNA can be further purified by precipitation with 1/3 volume of 7.5 M ammonium acetate and 2.5 volumes of ethanol.
10. Centrifuge as above, wash with 70% ethanol, and air dry. Resuspend in TE.

REFERENCES:

Coyer, J. A., Robertson, D. L. & Alberte, R. S. (1994). Genetic variablity within a population and between diploid/haploid tissue of Macrocystis pyrifera (Phaeophyceae). Journal of Phycology 30, 545-542.

Doyle, J. J. & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytoch. Bull. 19, 11-15.