Slides of mitotic chromosomes were prepared from imaginal discs of fourth instar larvae following published protocols3 (link),70 ,71 (link). BAC clones were obtained from the University of Liverpool19 or from a previously described BAC library72 (link). BACs were plated on agar plates (Thermo Fisher) and a single bacterial colony was used to grow an overnight bacterial culture in LB broth plates (Thermo Fisher) at 37 °C. DNA from the BACs was extracted using Sigma PhasePrep TM BAC DNA Kit (Sigma-Aldrich, NA-0100). BAC DNA for hybridization was labelled by nick translation with Cy3-, Cy5-dUTP (Enzo Life Sciences) or Fluorescein 12-dUTP (Thermo Fisher). Chromosomes were counterstained with DAPI in Prolong Gold Antifade (Thermo Fisher). Slides were analysed using a Zeiss LSM 880 Laser Scanning Microscope at 1,000× magnification. We note that localization of the M-locus to 1p11 is supported by both FISH and genomic analyses, but is contrary to a previously published placement at 1q2117 (link).
Agar BacterialChromosomes Clones Cy5 dutpDapi Fish Fluorescein 12 dutp GenomicGold HybridizationImaginal discs Larvae Laser scanning microscope M locus
Corresponding Organization : Rockefeller University
Other organizations :
Baylor College of Medicine, National Human Genome Research Institute, National Institutes of Health, California Institute of Technology, Columbia University, Neural Stem Cell Institute, Broad Institute, Princeton University, Liverpool School of Tropical Medicine, Southern Medical University, Purdue University West Lafayette, University College London, Colorado State University, National Center for Biotechnology Information, Yale University, Armed Forces Research Institute of Medical Science, QIMR Berghofer Medical Research Institute, Institut Pasteur, Department of Genomes & Genetics, Oxford Brookes University, University of California, Riverside, Rice University, Virginia Tech, University of Liverpool, University of California, San Diego, Texas A&M University, University of Illinois Urbana-Champaign, Harvard University
Preparation of slides of mitotic chromosomes from imaginal discs of fourth instar larvae
dependent variables
Localization of the M-locus
control variables
Protocols for preparing slides of mitotic chromosomes from imaginal discs of fourth instar larvae
controls
Positive control: None specified
Negative control: None specified
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