Mitotic Chromosome Preparation and FISH Analyses

Slides of mitotic chromosomes were prepared from imaginal discs of 4^th instar larvae following published protocols^{3 (link),70 (link),71 (link)}. BAC clones were obtained from the University of Liverpool^{19 (link)} or from a previously described BAC library^{72 (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^oC. DNA from the BACs was extracted using Sigma PhasePrep TM BAC DNA Kit (Sigma-Aldrich, catalogue #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 1000× 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 1q21^{17 (link)}.

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Matthews B.J., Dudchenko O., Kingan S.B., Koren S., Antoshechkin I., Crawford J.E., Glassford W.J., Herre M., Redmond S.N., Rose N.H., Weedall G.D., Wu Y., Batra S.S., Brito-Sierra C.A., Buckingham S.D., Campbell C.L., Chan S., Cox E., Evans B.R., Fansiri T., Filipović I., Fontaine A., Gloria-Soria A., Hall R., Joardar V.S., Jones A.K., Kay R.G., Kodali V.K., Lee J., Lycett G.J., Mitchell S.N., Muehling J., Murphy M.R., Omer A.D., Partridge F.A., Peluso P., Aiden A.P., Ramasamy V., Rašić G., Roy S., Saavedra-Rodriguez K., Sharan S., Sharma A., Smith M.L., Turner J., Weakley A.M., Zhao Z., Akbari O.S., Black WC I.V., Cao H., Darby A.C., Hill C.A., Johnston J.S., Murphy T.D., Raikhel A.S., Sattelle D.B., Sharakhov I.V., White B.J., Zhao L., Aiden E.L., Mann R.S., Lambrechts L., Powell J.R., Sharakhova M.V., Tu Z., Robertson H.M., McBride C.S., Hastie A.R., Korlach J., Neafsey D.E., Phillippy A.M, & Vosshall L.B. (2018). Improved reference genome of Aedes aegypti informs arbovirus vector control. Nature, 563(7732), 501-507.

Publication 2018

LB broth plates Sigma PhasePrep TM BAC DNA Kit Fluorescein 12-dUTP Prolong Gold Antifade LSM 880 Laser Scanning Microscope

Agar Bacterial Chromosomes Clones Cy5 dutp Dapi Fish Fluorescein 12 dutp Genomic Gold Hybridization Imaginal discs Larvae Laser scanning microscope M locus

Corresponding Organization : Rockefeller University

Other organizations : Baylor College of Medicine, Pacific Biosciences (United States), 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, BioNano Genomics (United States), 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

Top 5 similar protocols

Variable analysis

independent variables

Preparation of slides of mitotic chromosomes from imaginal discs of 4th instar larvae

dependent variables

Localization of the M-locus to 1p11

control variables

Preparation of slides of mitotic chromosomes from imaginal discs of 4th instar larvae following published protocols
Extraction of DNA from BACs using Sigma PhasePrep TM BAC DNA Kit
Labeling of BAC DNA for hybridization by nick translation with Cy3-, Cy5-dUTP or Fluorescein 12-dUTP
Counterstaining of chromosomes with DAPI in Prolong Gold Antifade
Analysis of slides using a Zeiss LSM 880 Laser Scanning Microscope at 1000× magnification

positive controls

Genomic analyses supporting the localization of the M-locus to 1p11

negative controls

Previously published placement of the M-locus at 1q21

Annotations

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