Isolation and Purification of Nuclear Dna from Excised Cotyledons of Cucurbita Pepo

High molecular weight genomic DNA was isolated from excised marrow cotyledons (Cucurbita pepo L., zucchini) using high concentration of ethidium bromide (400 μg.ml–1) in the grinding buffer and phenol deproteinization of the crude nuclear pellet. This preparation of DNA was free of proteins but contained polysaccharide contaminations which did not allow complete digestion with a number of restriction enzymes. Further purification of nuclear DNA was obtained by adsorbtion on Celite column and elimination of the polysaccharide contaminants. The UV absorbtion spectra of Celitepurified DNA showed that the ratio A260/A280 remained almost unchanged while the ratio A260/A230 significantly increased. As judged from the electrophoretic pattern of DNA restriction fragments in agarose gel as well as from the autoradioraphs after hybridization with 32P-labelled rDNA probe, Celite-purified DNA was completely digested with commonly used restriction enzymes Eco RI, Hind III, Bam HI. Thus, the purified DNA from excised cotyledons served for analysis of the methylation pattern of rDNA unit with methylation sensitive isoschizomers Msp I and Hpa II.