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13. SUPPLEMENTARY MATERIALS
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ANNEX 13.4. DNA ISOLATION
A modified version of CTAB method (Murray and Thompson 1980) was used for DNA
isolation. In this procedure nucleic acids form soluble complexes with a detergent CTAB
(Cetyltrimethylammonium bromide) in the presence of high concentration of NaCl. The complex
nucleic acids-CTAB is extracted from plant material with a chloroform and isoamyl alcohol
mixture. Polysaccharides, phenols and the others are removed by the precipitation of the
nucleic acid-CTAB complex in environment of low salt concentration. In these conditions the
complexes CTAB-nucleic acids form pellets while contaminants stay in a supernatant. CTAB
is removed from pellets by dissolving the CTAB-nucleic acid complex in a high concentration
of salt (NaCl or CsCl) and precipitation of nucleic acids with ethanol or isopropanol.
The leaves for DNA isolation were collected from plants growing in the field approximately
a month after the autumn cut in 2002 and 2003. About 4-5 g of leaves per a plant was
packed in a plastic, marked bag, sealed and kept in a cool place up to the moment they were
transferred to the laboratory. Then they were frozen and stored in -30 o C until use. DNA was
isolated from about 1 g of leaves. Prior to DNA isolation, all leaves were washed in deionized
water and surface sterilized with 96% ethanol. Then they were ground into a fine powder
in liquid nitrogen and thoroughly mixed with 3 ml of pre-warm CTAB isolation buffer (Table
13.4.1). The mixture was incubated for 60 min. at 55 o C with occasional shaking. Afterwards,
nucleic acids were extracted three times with chloroform and isoamyl alcohol by a ratio 24:1,
precipitated with the CTAB precipitation buffer (Table 13.4.1) for 24 h at room temperature
and dissolved in 1 ml of 1 M cesium chloride for 30 min. at 37 o C. After the subsequent precipitation
with 2.5 volume of ethanol, RNA was removed with RNA-ase at a final concentration
200 g/ml (Table 13.4.2). The DNA was then precipitated with 96% ethanol and the pellet was
washed with 70% ethanol and dissolved in deionized water. The quality of DNA was verified
on 1% (w/v) agarose containing 0.5 g/ml ethidium bromide and separated in 1 x TBE buffer
(Tris-Borate-EDTA; Sambrook et al. 1989) at 100 V constant power. The purity of DNA was
assessed spectrophotometrically and averaged 95-98%. Finally the aliquots were prepared at
a final concentration 500, 750 or 1000 ng/l. The DNA was stored at -30 o C until use.