Difference between revisions of "DNA Extraction Schizophyllum commune CTAB-PEG"

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# Add 200 μL of 96% ethanol and 88 μL of 5 M [[#Potassium acetate 5M CH3COOK | Potassium acetate (CH<sub>3</sub>COOK)]] 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.  
 
# Add 200 μL of 96% ethanol and 88 μL of 5 M [[#Potassium acetate 5M CH3COOK | Potassium acetate (CH<sub>3</sub>COOK)]] 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.  
 
# 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.  
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# Add 800 μL of [[#chloroform:isoamyl alcohol (24:1 v/v) | 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.  
 
# 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 163 μL of [[5M NaCl]] and mix them. Make sure that the terminal concentration of NaCl is 0.7 M.  
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== Reagents and solutions ==
 
== Reagents and solutions ==
In this study, buffers and solutions used for DNA isolation from mycelium of Schizophyllum commune 15R-5-F01 are as follows:
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Buffers and solutions used for DNA isolation from mycelium of ''Schizophyllum commune''.
 +
 
 
=== 4% CTAB extraction buffer:===
 
=== 4% CTAB extraction buffer:===
 
For 100 ml:
 
For 100 ml:
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===1.4 M NaCl===
 
===1.4 M NaCl===
For 100 ml:
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For 100 ml
 
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Dissolve 8.2 g NaCl in demiwater
dissolve 58.44 g/mol
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Sterilize by autoclaving
  
 
===Potassium acetate 5M CH3COOK===
 
===Potassium acetate 5M CH3COOK===
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===CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl)===
 
===CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl)===
===ethanol (96 and 70% in H2O, v/v)===
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For 100 ml:
RNase (Sangon Biotech, China)
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* 10 g CTAB
20% PEG 8000 solution (1.2 M NaCl)
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* 4.1 g NaCl
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* 5ml 1M Tris-HCl, pH 8.0
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* 2ml 1M EDTA, pH 8.0
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* fill to 100ml with demiwater
 +
 
 +
===20% PEG 8000 solution (1.2 M NaCl)===
 +
For 100 ml:
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* 20 g PEG-8000
 +
* 7 g NaCl

Revision as of 16:26, 17 June 2020

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 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 (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.
  8. Add 200 μL of 96% ethanol and 88 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. Store the tubes for 30 min at 4°C in the fridge.
  9. Allow the tubes to warm up to room temperature for ~3 min.
  10. 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.
  11. 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.
  12. Add 163 μL of 5M NaCl and mix them. Make sure that the terminal concentration of NaCl is 0.7 M.
  13. 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).
  14. 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.
  15. 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.
  16. 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.
  17. Repeat steps XXXXX
  18. Transfer the upper aqueous phase to a fresh microfuge tube.
  19. 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.
  20. Precipitate the nucleic acids by centrifugation at 10,000 xg for 10 min at 4°C in the Cooling centrifuge.
  21. Remove the supernatant by aspiration.
  22. Rinse the pellet with 200 μL of 70% ethanol.
  23. Centrifuge the tubes at 10,000 xg for 1 min at room temperature in a microfuge.
  24. Rinse the pellet once more with 200 μL 70% ethanol.
  25. Centrifuge again at 10,000 xg for 1 min at room temperature.
  26. Combine the precipitates from all tubes into 1–2 tubes. It can increase the concentration of DNA.
  27. 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

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

1.4 M NaCl

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

Potassium acetate 5M CH3COOK

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

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

20% PEG 8000 solution (1.2 M NaCl)

For 100 ml:

  • 20 g PEG-8000
  • 7 g NaCl