DNA Extraction Schizophyllum commune CTAB-PEG

Extraction protocol for high-quality, high-molecular-weight genomic DNA

This protocol is based on paper 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 errata in that protocol can be found here.

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 4.5 mL of [#4% CTAB extraction buffer (pH 8.0)] to the mortar.
4. Incubate the digest for 40–60 min at 65°C. The mortar should be sealed to prevent contamination during digestion.
5. Mix the digest well.
6. Transfer 4 mL of the lysis suspension to a fresh 15ml Falcon tube.
7. Preheated CTAB/NaCl solution to 65°С. This step cannot be omitted. Without heating up the CTAB/NaCl, the solution is sticky, and cannot be pipetted easily.
8. Add 1 mL of 96% ethanol and 440 μL of 5 M Potassium acetate (CH₃COOK) to each tube (ethanol:CH₃COOK = 25:11, v/v), and mix contents of the tube by vortexing for 1 min.
9. Incubate the tubes for 30 min at 4°C in the fridge.
10. Allow the tubes to warm up to room temperature for ~3 min.
11. Add 4 mL chloroform:isoamyl alcohol (24:1 v/v) to each tube, and mix the organic and aqueous phases by vortexing for 1 minute.
12. Separate the mixture into three phases by centrifuging at 10,000 xg for 15 min at 20°C.
13. Transfer 4.5 ml of the upper aqueous phase to a fresh tube.
14. Add 733 μL of 5M NaCl and mix by inversion. The final concentration NaCl should be 0.7 M.
15. Add 594 μL of CTAB/NaCl solution (pH 8.0, preheated for >10min at step 4 ) and mix well by inverting multiple times.
16. Incubate the tubes for 15 min at 65°C.
17. Add 5 μL of DNase-free RNase A (100 mg/ml).
18. Incubate the mixture for 30 min at 50°C.
19. 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.
20. 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.
21. Repeat steps XXXXX
22. Transfer the upper aqueous phase to a fresh microfuge tube.
23. 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.
24. Precipitate the nucleic acids by centrifugation at 10,000 xg for 10 min at 4°C in the Cooling centrifuge.
25. Remove the supernatant by aspiration.
26. Rinse the pellet with 200 μL of 70% ethanol.
27. Centrifuge the tubes at 10,000 xg for 1 min at room temperature in a microfuge.
28. Rinse the pellet once more with 200 μL 70% ethanol.
29. Centrifuge again at 10,000 xg for 1 min at room temperature.
30. Combine the precipitates from all tubes into 1–2 tubes. It can increase the concentration of DNA.
31. 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.

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Reagents and solutions

Buffers and solutions used for DNA isolation from mycelium of Schizophyllum commune.

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

Store at RT in drawer under fume hood Note that CTAB needs to be disposed of properly.

CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl)

For 100 ml:

• 10 g CTAB
• 4.1 g NaCl
• 5ml 1M Tris-HCl, pH 8.0
• 2ml 1M EDTA, pH 8.0
• fill to 100ml with demiwater

Store at RT in drawer under fume hood Note that CTAB needs to be disposed of properly.

1.4 M NaCl

For 100 ml Dissolve 8.2 g NaCl in 100 ml demiwater Sterilize by autoclaving

5 M NaCl

For 100 ml Dissolve 29.2 g NaCl in 100 ml demiwater Sterilize by autoclaving and store at RT

Potassium acetate 5M CH3COOK

For 100 ml Dissolve 49.1 g CH3COOK in 100 ml demiwater Sterilize by autoclaving and store at RT

chloroform:isoamyl alcohol (24:1 v/v)

In a fume hood, mix in a 250 ml Greiner bottle:

• 96 ml Chloroform
• 4 ml Isoamyl alcohol

Store at RT in drawer under fume hood Note that chloroform:isoamyl alcohol needs to be disposed of properly.

20% PEG 8000 solution (1.2 M NaCl)

For 100 ml:

• 20 g PEG-8000
• 7 g NaCl
• fill to 100ml with demiwater

Store at 4°C