Difference between revisions of "DNA Extraction Schizophyllum commune CTAB-PEG"
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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. | + | # 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. | + | # 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. | + | # Add 4 mL of [4% CTAB extraction buffer (pH 8.0)] to the mortar. |
− | #Mix the digest well. | + | # Incubate the digest for 40–60 min at 65°C. The mortar should be sealed to prevent contamination during digestion. |
− | # | + | # Mix the digest well. |
− | #Add 200 μL of ethanol and 88 μL of 5 M | + | # Transfer 4 mL of the lysis suspension to a fresh 15ml Falcon tube. |
− | #Allow the tubes to warm up | + | # 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. |
− | #Separate the mixture into three phases by centrifuging at | + | # Add 200 μL of 96% ethanol and 88 μL of 5 M [[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 163 μL of 5M NaCl and mix them. Make sure that the terminal concentration of NaCl is 0.7 M. | + | # Allow the tubes to warm up to room temperature for ~3 min. |
− | #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). | + | # 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 H<sub>2</sub>O. | ||
− | 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: | 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% CTAB extraction buffer:=== |
4% w/v CTAB | 4% w/v CTAB | ||
50 mM Tris-HCl, pH 8.0 | 50 mM Tris-HCl, pH 8.0 | ||
20 mM EDTA, pH 8.0 | 20 mM EDTA, pH 8.0 | ||
− | 1.4 M NaCl | + | ===1.4 M NaCl=== |
+ | |||
+ | ===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)=== | |
− | |||
− | CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl) | ||
− | ethanol (96 and 70% in H2O, v/v) | ||
RNase (Sangon Biotech, China) | RNase (Sangon Biotech, China) | ||
20% PEG 8000 solution (1.2 M NaCl) | 20% PEG 8000 solution (1.2 M NaCl) |
Revision as of 16:09, 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.
- 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 (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.
- 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:
4% w/v CTAB 50 mM Tris-HCl, pH 8.0 20 mM EDTA, pH 8.0
1.4 M NaCl
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)