. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. CHROMOSOMAL REARRANGEMENT IN PECTINIDAE 249 and a final 5-min extension at 72 °C. PCR products were evaluated on 1% (w:v) agarose gels and visualized by I /ng/ml ethidium bromide staining and ultraviolet illumina- tion. Fluorescence in situ hybridization FISH was carried out according to Guo and Allen (1997a), with slight modifications. Chromosomes were pretreated by incubating the slides in 2X SSC ( M sodium chloride, M sodium citrate, pH ) for 30 min at 37 °C; dehydrated in 70%, 80%, and 95% ethanol for 2 min ea
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. CHROMOSOMAL REARRANGEMENT IN PECTINIDAE 249 and a final 5-min extension at 72 °C. PCR products were evaluated on 1% (w:v) agarose gels and visualized by I /ng/ml ethidium bromide staining and ultraviolet illumina- tion. Fluorescence in situ hybridization FISH was carried out according to Guo and Allen (1997a), with slight modifications. Chromosomes were pretreated by incubating the slides in 2X SSC ( M sodium chloride, M sodium citrate, pH ) for 30 min at 37 °C; dehydrated in 70%, 80%, and 95% ethanol for 2 min each; and air-dried. Chromosomes were dena- tured in 70% formamide in 2x SSC (pH ) at 72 °C for 2 min and then dehydrated in a cold ethanol series (70%, 80%, and 95%) and air-dried. The labeled probes were diluted in hybridization solution, 65% formamide in 2X SSC, at a ratio of 1:15; denatured at 72 °C for 5 min; and placed immediately on ice. Probe mixture (15-20 /n,l) was applied to each denatured slide, covered with a glass coverslip, and sealed with rubber cement. For dual-hy- bridization of two probes on the same metaphase, two probes were denatured and mixed before application. Slides were then incubated at 37 °C in a humidity incu- bator overnight for hybridization. After hybridization, the coverslips were removed and the slides were washed twice in 2x SSC at 72 °C for 5 min each time, with 1 X PBT ( M NaH2PO4, BSA, Tween-20, pH ) at room temperature for 2 min. The digoxigenin- labeled probes were detected with fluorescein-labeled anti-digoxigenin antibody. Chromosomes were counter- stained with jug/ml of propidium iodide (PI) in anti- fade solution (Vector Laboratories) and viewed under a Nikon epifluorescence microscope. FISH signals and karyotype were captured using a 3CCD camera and an- alyzed using the Image-Pro Plus software. Chromo- somes were classified according to the criteria defined by Levan et al. (1964). Results Probe quality
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
