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== Extraction protocol for high-quality, high-molecular-weight genomic DNA == This protocol is based on paper [https://doi.org/10.1134/S0026893318040088 Huang et al. “CTAB-PEG DNA Extraction from Fungi with High Contents of Polysaccharides.” Molecular Biology 52, no. 4 (July 1, 2018): 621–28.] the [https://link.springer.com/article/10.1134/S0026893319080016 errata in that protocol can be found here]. # Prepare fresh mycelia from [[IPDA]] plates. Dry the mycelia on filter paper. The recommendated amount of sample is 0.09–0.12 g (dry weight) for each operation. # Freeze the dried mycelium in [[liquid nitrogen]] and grind it to a powder in a mortar prechilled with liquid nitrogen. # Add 4 mL of [#4% CTAB extraction buffer (pH 8.0)] to the mortar. # Incubate the digest for 40–60 min at 65°C. The mortar should be sealed to prevent contamination during digestion. # Mix the digest well. # Transfer 4 mL of the lysis suspension to a fresh 15ml Falcon tube. # Preheated [[#CTAB/NaCl solution (pH 8.0)]] to 65°С. This step cannot be omitted. Without heating up the CTAB/NaCl, the solution is sticky, and cannot be pipetted easily. # Add 200 μL of 96% ethanol and 88 μL of 5 M [[#Potassium acetate 5M CH3COOK | Potassium acetate (CH₃COOK)]] to each tube (ethanol:CH<sub>3<sub>COOK = 25:11, v/v), and mix contents of the tube by vortexing for 1 min. Store the tubes for 30 min at 4°C in the fridge. # Allow the tubes to warm up to room temperature for ~3 min. # Add 800 μL of [[#chloroform:isoamyl alcohol (24:1 v/v)]] to each tube, and mix the organic and aqueous phases by vortexing for 1 minute. # Separate the mixture into three phases by centrifuging at 10,000 xg for 15 min at 20°C. Transfer the upper aqueous phase to the fresh tubes. Each tube must contain only 1 mL of the upper aqueous phase. # Add 163 μL of [[5M NaCl]] and mix them. Make sure that the terminal concentration of NaCl is 0.7 M. # Add 132 μL of CTAB/NaCl solution(pH 8.0, preheated at step 4) and mix well, then incubate the tubes for 15 min at 65°C. CTAB/NaCl solution must be preheated to 65°C for a while (>10 min). # Allow the tubes to cool to room temperature for 3 min. Add 5 μL of DNase-free RNase A (100 mg/ml). Incubate the mixture for 30 min at 50°C. # Transfer each 900 μL of the mixture to a fresh microfuge tube. Add an equal volume of chloroform: isoamyl alcohol (24 : 1, v/v) to each tube, and mix the organic and aqueous phases by vortexing for 1 min. # Separate the mixture into three phases by centrifuging at 10,000 xg for 15 min at 20°C. Transfer the upper aqueous phase to fresh tubes. # Repeat steps XXXXX # Transfer the upper aqueous phase to a fresh microfuge tube. # Collect the DNA by precipitation with an equal volume of 20% PEG-8000 (pH 8.0, contains 20% PEG-8000 w/v, 1.2M NaCl) for 1–2 h at –20°C or at 4°C overnight. # Precipitate the nucleic acids by centrifugation at 10,000 xg for 10 min at 4°C in the [[Cooling centrifuge]]. # Remove the supernatant by aspiration. # Rinse the pellet with 200 μL of 70% ethanol. # Centrifuge the tubes at 10,000 xg for 1 min at room temperature in a microfuge. # Rinse the pellet once more with 200 μL 70% ethanol. # Centrifuge again at 10,000 xg for 1 min at room temperature. # Combine the precipitates from all tubes into 1–2 tubes. It can increase the concentration of DNA. # Remove the supernatant by aspiration and allow the pellet to dry in the air for 1–2 min. Redissolve the pellet in 20–40 μL of DNase-free H₂O. ---- == Reagents and solutions == In this study, buffers and solutions used for DNA isolation from mycelium of Schizophyllum commune 15R-5-F01 are as follows: === 4% CTAB extraction buffer:=== For 100 ml: *4g CTAB *5ml 1M Tris-HCl, pH 8.0 *2ml 1M EDTA, pH 8.0 *fill to 100ml with demiwater ===1.4 M NaCl=== For 100 ml: dissolve 58.44 g/mol ===Potassium acetate 5M CH3COOK=== ===chloroform:isoamyl alcohol (24:1 v/v)=== ===CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl)=== ===ethanol (96 and 70% in H2O, v/v)=== RNase (Sangon Biotech, China) 20% PEG 8000 solution (1.2 M NaCl)

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