Please cite as: CSH Protocols; 2007; doi:10.1101/pdb.prot4730

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Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes

Stefan Müller1, Michaela Neusser, Daniela Köhler, and Marion Cremer

Ludwig-Maximilians University Munich, Department Biology II, AG Thomas Cremer (Chair of Anthropology and Human Genetics), 82152 Martinsried-Planegg, Germany

1Corresponding author (S.Mueller@lrz.uni-muenchen.de)


INTRODUCTION

DNA probes for fluorescence in situ hybridization (FISH) can be generated and labeled by various methods. This protocol describes the conjugation of dUTPs with haptens or fluorochromes, as well as the generation and labeling of DNA probes using those modified dUTPs. Sources of probe DNA include genomic DNA, DNA from flow-sorted chromosomes, bacterial artificial chromosomes (BACs), and cosmids. DNA amplification and labeling procedures involving degenerate oligonucleotide-primed PCR (DOP-PCR) and multiple displacement amplification (MDA) are provided. Advice is given for setting up complex probe pools, such as those containing large pools of BAC probes. Also included is a method for probe precipitation and preparation of a hybridization mix ready to be used for 3D fluorescence in situ hybridization (FISH) experiments.


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