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J Vis Exp. 2013 Aug 15;(78). doi: 10.3791/50587.

Robust 3D DNA FISH using directly labeled probes.

Author information

1
Nuclear Dynamics Programme, The Babraham Institute. daniel.bolland@babraham.ac.uk

Abstract

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

PMID:
23978815
PMCID:
PMC3846859
DOI:
10.3791/50587
[Indexed for MEDLINE]
Free PMC Article

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