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Isolation of High Molecular Length DNA

Alfalfa, Pea, Rice, Sorghum, Soybean, and Spinach

^a B.M. Sutherland, Biology Dep., Brookhaven National Laboratory, Upton, NY 11973-5000
^b Kyoto Univ., Gokanoshi, Uji, Kyoto 611-0011, Japan
^c Institute of Genetic Ecology, Tohoku Univ., Sendai, Japan
^d Dep. of Pathology, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115
^e Dep. of Natural Resource Science and Landscape Architecture, Univ. of Maryland, College Park MD 20742
^f Argonne National Laboratory, Argonne, IL 60439
^g Dep. of Biology, Toho Univ. School of Medicine, Tokyo, Japan

View larger version (68K): [in a new window]   Fig. 1. Three alkaline agarose gels showing DNA of unirradiated pea seedling leaves resulting from different isolation procedures. A. Lanes 1 and 2, Trial Procedure I (phenol, NaClO4); Lanes 3 and 4, Trial Procedure II (phenol; chloroform:isoamyl alcohol); Lanes 5 and 6, Trial Procedure III (potassium acetate); Lane 7, Trial Procedure IV (phenol:chloroform:isoamyl alcohol, isopropanol precipitation); Lane 8, Trial Procedure V (phenol:chloroform:isoamyl alcohol, ethanol precipitation). B. Lane 9, protoplast production. C. Lane 10, agarose plug preparation. Panels A, B, and C are independent gels; the panels are aligned at the positions of the (48.5 kb) and T7 (39.9 kb) markers. In panel A, Lanes 3 and 4, as well as 7, 8, and M are longer (photographic) exposures of the same gel, since DNA in these lanes was very faint. Marker (M) lanes in gels A and B contained (48.5 kb) and T7 (39.9 kb) DNAs, as well as a HindIII digest of (23.1, 9.4, 6.6, 4.4, 2.3, 2 and 0.56 kb); the marker lane on Gel C also contained T4 DNA (170 kb)

View larger version (37K): [in a new window]   Fig. 2. Sections of alkaline agarose gels of DNA from higher plants, and molecular length marker DNAs. Plants were unirradiated, except for F, field-grown soybeans, which were exposed to ambient sunlight. Panel A: Alfalfa—G, bacteriophage G; M + T, bacteriophage T4, , and HindIII digest of ; A, alfalfa. Panel B: Spinach—S, spinach; T, T4; M, , and HindIII digest of . Panel C: Rice—H, H. wingei chromosomes; T, T4; M, and HindIII digest of ; R, rice. Panel D. Sorghum—H, H. wingei; T, T4; M, and HindIII digest of ; Sgh, dark-grown sorghum seedlings. Panel E: Soybean—Sb, soybean seedlings grown in environmental chamber; M, , and HindIII digest of ; G + T, bacteriophages G and T4. Panel F: Field grown soybean seedlings—Sb, soybean; G, bacteriophage G, M + T, T4 plus , and HindIII digest of . Unidirectional pulsed field electrophoresis was used, except for rice, for which static field electrophoresis was used (50 V, 40 min). Each panel contains lanes from one gel, juxtaposed for clarity if necessary; the intensity of marker H in Panel C was increased electronically for visibility in the photograph

View larger version (32K): [in a new window]   Fig. 3. A. Electronic image of DNA from rice seedlings exposed to 0.9, 1.8, 2.7, 3.6, and 0 kJ/m2 of UVB radiation. Samples are paired; the first (-) was incubated with buffer alone, while the companion sample (+) was incubated with UV endonuclease. Molecular length standard DNAs are bacteriophages G (750 kb) and T4 (170 kb); marker Lane M contains and a HindIII digest of . B. Cyclobutyl pyrimidine dimer induction in DNA in rice seedlings as a function of exposure to broad spectrum UV radiation from an FS–20 lamp. Data were obtained from gels such as in Panel A. Small symbols, individual samples (electrophoresed on replicate independent gels, data from each gel shown as one symbol—filled circles, filled triangles, filled diamond, or filled inverted triangles) from the same plant material; large symbols, averages; error bars, standard errors. In some cases the standard error is less than the size of the symbol for the average