| Abstract: |
Fluorescence in situ hybridization (FISH) provides a quick means of placing almost any recombinant DNA clone onto a physical map. The shift to large-insert, single-copy plasmids such as PACs and BACs has substantially reduced the problem of clone chimerism. However, large-scale genome sequencing projects are revealing significant levels of regional homology that can confound mapping analyses at all levels. Whole clone DNA preparations are usually the best material for nick translation. Bacterial clone DNA isolated by standard alkaline lyses should be satisfactory. Different grades of DNase I have different levels of activity. New working solutions should be titrated to determine the best incubation time. Ethanol precipitation enables the labeled probe to be concentrated so that it can be used without further precipitation prior to hybridization. This ensures more consistent results from one experiment to another. Large insert clones can be mapped efficiently using direct labeling with fluorochrome-conjugated dUTPs. © 2006 Copyright © 2006 Elsevier Inc. All rights reserved. |
