Phenol chloroform isoamyl alcohol 25 24 1

Five to 6 frozen adults were ground to powder with a mortar and pestle under liquid nitrogen. Powder was dissolved in 4 mL fresh STE buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, pH 8). Two hundred μL 10 % SDS and 8 μL RNase A (Cat# R4642-250MG Sigma Aldrich St. Louis, Missouri) were added and samples were incubated at 56 °C. After 30 min, Proteinase K (Cat# P2308-100MG Sigma Aldrich) was added to 100 μg/mL and the sample was incubated overnight at 56 °C. Three mL phenol:chloroform:isoamyl alcohol [25:24:1] (Cat#P2069, Sigma) was added and samples were rotated 10 min at 12 RPM at 22 °C. Samples were then centrifuged 10 min at 1000 g at 4 °C. The aqueous layer was transferred to a new tube and the extraction was repeated. One tenth volume 3 M sodium acetate, pH 5.2 and 2 volumes cold 100 % ethanol were added and mixed. The samples were incubated at -20 °C for 1 h and centrifuged 30 min at 7200 g at 4 °C. The supernatant was removed from the pellet, which was washed with 1 mL cold 75 % ethanol. The pellet was air-dried 10 min before resuspension in 50–100 μL TE Buffer. To determine genomic sequence flanking the transgene insertion, inverse PCR was performed with MboI, TaqαI, and MspI-digested genomic DNA templates as described previously [48 (link), 71 (link)].

Five to 6 frozen head/thoraxes were ground to powder with a mortar and pestle under liquid nitrogen. Powder was dissolved in 4 mL STE buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, pH 8). Two hundred μL 10% SDS and 8μL RNase A (Cat# R4642 Sigma Aldrich St. Louis, Missouri) were added and samples were incubated at 56°C. After 30 min, Proteinase K (Cat# P2308 Sigma Aldrich) was added to 100 μg/mL and the sample was incubated overnight at 56°C. Three mL phenol:chloroform:isoamyl alcohol [25:24:1] (Cat#P2069, Sigma) was added and samples were rotated 10 min at room temperature (RT). Samples were then centrifuged 10 min at 3000 RPM at 4°C. The aqueous layer was transferred to a new tube. The extraction was repeated. One tenth volume 3M NaAcetate, pH 5.2 and 2 volumes cold 100% ethanol were added and the samples were inverted 2–3 times. The samples were incubated at -20°C for 1 h and centrifuged 30 min at 6000 RPM at 4°C. The supernatant was removed from the pellet and 1 mL cold 75% ethanol was added. The samples were centrifuged 10 min at 6000 RPM at 4°C. The supernatant was removed from the pellet and it was allowed to air dry 10 min. The last of the supernatant was removed and the pellet was allowed to air dry 10 additional minutes before being resuspended in 50–100 μL TE Buffer.

Using stock-cultures stored at −80 °C, cultures were prepared in YPD medium at 28 °C. After growth until the stationary phase, cells were harvested by centrifugation (9150× g for 4 min at 4 °C), washed with 1 mL of sterile water and then re-suspended in 200 µL of extraction buffer (SDS 1% w/v, Triton × 100 2% w/v, NaCl 100 mM, Tris 10 mM, EDTA 1 mM, pH = 8), with 0.3 g glass beads (0.5 mm in diameter; Scientific Industries) and 60 µL phenol:chloroform:isoamyl alcohol 25:24:1 (Sigma). The cells were broken for 3 × 45 s at 6500 rpm with a 60-s interval using a Precellys 24-Dual Homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). After the addition of 200 µL of Tris EDTA buffer (Tris 10 mM, EDTA 1 mM, pH 8), the cultures were centrifuged at 18,200× g for 10 min at 4 °C. The aqueous phase was collected and the DNA was precipitated with 1 mL of 100% ethanol and then centrifuged at 18,200× g for 10 min at 20 °C. The pellet was washed with 70% ethanol and centrifuged at 18,200× g for 5 min at 20 °C. Then, the pellet was dried and resuspended in sterile water. Finally, the samples were stored at −20 °C. The concentration and purity of DNA was measured by Infinite M200 Pro NanoQuant (TECAN, Lyon, France).