DNA Extraction Schizophyllum commune CTAB-PEG
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- 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. Then transfer each 800 μL of the digest to a fresh microfuge tube.
- Incubate in preheated to 65°С CTAB/NaCl solution (pH 8.0). This step cannot be omitted. Without CTAB/NaCl the solution is sticky, and cannot be sampled easily.
- Add 200 μL of ethanol and 88 μL of 5 M CH3COOK to each tube (ethanol: CH3COOK = 25 : 0.11, 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 at room temperature for 3 min. Then 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 12000 rpm (10000 g in a Sorvall SS-34 rotor) 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).
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: 4% w/v CTAB 50 mM Tris-HCl, pH 8.0 20 mM EDTA, pH 8.0
1.4 M NaCl
5M CH3COOK 10 μM proteinase K 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)