Fluorescent in situ hybridization of BACs to metaphase chromosome spreads were performed as previously described74 ,75 (link). A Qiagen Large Construct kit (Qiagen Science, 12462) was used to extract bacterial artificial chromosome (BAC) DNA for E24C3 and E12A6, previously associated with sex29 (link). Probes for in situ hybridization were labeled by nick-translation using direct fluorophores Cyanine 3-dUTP (Enzo Life Sciences, ENZ-42501) or Fluorescein-12-dUTP (Thermo Scientific, R0101) as described previously74 and hybridization of BAC probes was performed as previously described for axolotl chromosomes40 (link).
Phenol-chloroform extraction in 1.2X SSC was used to isolate repetitive DNA fractions from female salamander tissue76 . DNA was denatured for 5 minutes at 120 °C, re-associated at 60 °C for 1 hour to obtain Cot DNA. Microtubes containing the DNA were placed on ice for 2 minutes, then transferred to a bead bath at 42 °C for 1 hour with 5X S1 nuclease buffer and S1 nuclease for a concentration of 100 units per 1 mg DNA. DNA was precipitated with 0.1 volume of 3M sodium acetate and 1 volume isopropanol at room temperature, tubes were inverted several times and centrifuged at 14,000 rpm for 20 minutes at 4 °C. DNA was washed with 70% ethanol, centrifuged at 14,000 rpm for 10 minutes at 4 °C, air dried and solubilized in TE buffer.
Phenol-chloroform extraction in 1.2X SSC was used to isolate repetitive DNA fractions from female salamander tissue76 . DNA was denatured for 5 minutes at 120 °C, re-associated at 60 °C for 1 hour to obtain Cot DNA. Microtubes containing the DNA were placed on ice for 2 minutes, then transferred to a bead bath at 42 °C for 1 hour with 5X S1 nuclease buffer and S1 nuclease for a concentration of 100 units per 1 mg DNA. DNA was precipitated with 0.1 volume of 3M sodium acetate and 1 volume isopropanol at room temperature, tubes were inverted several times and centrifuged at 14,000 rpm for 20 minutes at 4 °C. DNA was washed with 70% ethanol, centrifuged at 14,000 rpm for 10 minutes at 4 °C, air dried and solubilized in TE buffer.
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