DNA Extraction Schizophyllum commune CTAB-PEG

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  1. 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.
  2. Freeze the dried mycelium in liquid nitrogen and grind it to a powder in a mortar prechilled with liquid nitrogen.
  3. 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.
  4. Mix the digest well. Then transfer each 800 μL of the digest to a fresh microfuge tube.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. Add 163 μL of 5M NaCl and mix them. Make sure that the terminal concentration of NaCl is 0.7 M.
  10. 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)