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Fig 5 (FISH guide)
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Legend Fig. 5. Size of labeled DNA fragments in hybridizations. In all images arrow indicate the 500 bp band in the 1 kb ladder. Arrowhead shows the strong fluorescence of FITC and rhodamine in the direct-labeled nucleotides used during the PCR labeling reaction. a) DNA fragments of probes labeled by nick translation have an average size around 200-300 bp. b) DOP-PCR amplification of a several chromosome painting probes. The fragments have a variety of sizes, some very large. Some of the amplified DNA was subjected to DNase digestion for 2 minutes (c) or 12 minutes (d). Undigested and digested DNA was used for FISH. In (e), DNA labeling was checked by nick translation. One microliter each from serial dilutions of three different biotin-labeled DNA probes were spotted on a small nylon membrane. The membrane was processed and detected using the colorimetric reaction of alkaline phosphatase onto the NBT/BCIP substrate, according to a standard protocol. A well labeled DNA sample should be visible even when diluted to 10pg/ul or lower. Images f, g, h are the DAPI counterstained version of images i, j, k, respectively. Undigested DNA did not stain the chromosomes , it "stopped" at the edges of the chromosomes (i) where, probably, loops of DNA protrude out of the chromosomal mass. Only repetitive sequences were probably hybridized, as no specific signals were visible. The rather large probe fragments were probably trapped in that position and were not washed away during the posthybridization steps. The DNase digested fragments yielded specific painting signals, but the signal to noise ratio was better with the 12 minutes digested fragments (k) than with the 2 minutes ones (j).
