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For further background suppression, the filter is prehybridized in these agents prior to addition of the probe. Background can also be of a more specific nature. ” There is a wide range of these elements dispersed throughout the genome of all eukaryotic organisms, and they vary m size and frequency. For example, on average, there is one Ah repeat every 5 kb in the human genome (3). This means that long probes prepared from genomrc DNA, such as whole h or cosmid clones, may hybridize to many genomic locations and produce a smear that masks the signal from singlecopy sequences.
The addition of sheared, denatured heterologous DNA to the hybridization buffer can reduce nonspecific hybrtdization, although some reduction in sensitrvtty may also be observed. 5. Hybridization can be carried out m either boxes, bags, or tubes provided there is sufficient buffer to allow adequate accessof probe to the blot. With larger blots (>50 cm2), or if using mimmal hybridization volumes m bags or tubes, the blots should be prewetted in 5X SSC. It 1salso possible to hybridize several blots in the same solutton, again providing there is adequate volume and circulation of buffer, A maximum of two blots should be hybridized together in a single bag, and when using more than one blot in a tube, it is important that the blots do not overlap one another.
Wash the membranes for 30 min at 65OC with at least 200 mL of washing solution and with good agitation. This step removes any probe that is not bound to the membrane. 7. Wash the membranes with buffer 1 for 5 min at room temperature (this and all subsequent washes are performed at room temperature and with vigorous agitation). This step removes the washing solution from the membranes and prepares the surface for the blocking agent. 8. Incubate the blots with 200 mL of blocking buffer for 30 min.