Run 1
High quality axolotl genomic DNA was prepared from freshly isolated liver and spleen of anindividual three-year-old adult D/D male. We first cooled down mortars and pestles by fillingwith liquid nitrogen, and then ground 500-750 g liver or 300-500 spleen tissues into a finepowder in liquid nitrogen. We added 30 ml of DNAzol reagent (ThermoFisher Scientific,10503027), or 30 ml of DNA extraction buffer (100 mM Tris-Cl (pH 8.0), 2 mM EDTA (pH8.0), 400 mM NaCl, freshly added 0.8% SDS and 0.3 mg/ml Proteinase K) into the liver orspleen tissue powder, respectively, transferred the cell lysate into a 50 ml Falcon tube andincubated at room temperature (liver) or 55C (spleen) for 1-3 hours. The cell lysate wascentrifuged at 10,000g for 30 minutes at 4C. We then carefully transferred the supernatantinto a new Falcon tube, added an equal volume of phenol/chloroform (Sigma, P2069) andgently rotated the tube for 15-30 minutes at 4C followed by centrifugation at 10,000 g for 30minutes at 4C. We carefully transferred the upper aqueous phase into a new 50 ml Falcontube and repeated once more the phenol/chloroform extraction followed by two chloroformextractions using similar procedures. We added 1/10 volume of 3M sodium acetate (pH 5.2)and 2.5 volumes of pure ethanol into the final recovered liquid, and gently rotated the tubeseveral times to precipitate the genomic DNA. We fished out the precipitated white cottonlike genomic DNA with a clean loop and washed it three times in a large volume of 70%ethanol. After the final wash, we pelleted down the DNA at 13,000g for 15 minutes anddiscarded the liquid. As a final step, we briefly air-dried the genomic DNA, dissolved it in TEbuffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for further analysis