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]. | ||
− | + | We were not as successful with this as hoped. We used the PEG-precipitation technique from this protocol and used it for a modified version of the [[DNA Extraction Phenol Chlorophorm S. commune|DNA Extraction Phenol Chlorophorm ''S. commune'']] protocol. | |
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− | + | [[DNA Extraction S. commune Phenol Chlorophorm PEG|USE THIS PROTOCOL "DNA Extraction S. commune Phenol Chlorophorm PEG" FOR DNA EXTRACTIONS]] | |
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− | 1.4 M NaCl | + | # 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 4.5 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 (10% w/v CTAB, 0.7 M NaCl) | 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. | ||
+ | # Add 1 mL of 96% ethanol and 440 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. | ||
+ | # Incubate the tubes for 30 min at 4°C in the fridge. | ||
+ | # Allow the tubes to warm up to room temperature for ~3 min. | ||
+ | # Add 4 mL [[#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 4.5 ml of the upper aqueous phase to a fresh tube. | ||
+ | # Add 733 μL of [[#Sodium chloride - 5M NaCl | 5M NaCl]] and mix by inversion. The final concentration NaCl should be 0.7 M. | ||
+ | # Add 594 μL of CTAB/NaCl solution (pH 8.0, preheated for >10min at step 4 ) and mix well by inverting multiple times. | ||
+ | # Incubate the tubes for 15 min at 65°C. | ||
+ | # Add 25 μL of DNase-free RNase A (100 mg/ml). | ||
+ | # Incubate the mixture for 30 min at 50°C. | ||
+ | # Add 5850 µl (an equal volume) of [[#Chloroform:isoamyl alcohol (24:1 v/v)|chloroform: isoamyl alcohol]] to the 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 a fresh tube. | ||
+ | # Add an equal volume of [[#Chloroform:isoamyl alcohol (24:1 v/v)|chloroform: isoamyl alcohol]] to the 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 a fresh tube. | ||
+ | # Add an equal volume of [[#20% PEG-8000 precipitation solution (with 1.2 M NaCl) | 20% PEG-8000, 1.2M NaCl]] and mix by inverting the tube multiple time. | ||
+ | # Collect the DNA by precipitation with 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 500 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 500 μL 70% ethanol. | ||
+ | # Centrifuge again at 10,000 xg for 1 min at room temperature. | ||
+ | # Remove the supernatant by aspiration | ||
+ | # Allow the pellet to dry in the air for 1–2 min. | ||
+ | # Elute the pellet in 40 μL of DNase-free H<sub>2</sub>O. | ||
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− | + | == Reagents and solutions == | |
− | + | Buffers and solutions used for DNA isolation from mycelium of ''Schizophyllum commune''. | |
− | CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl) | + | |
− | + | === 4% CTAB extraction buffer:=== | |
− | + | For 100 ml: | |
− | 20% PEG 8000 solution (1.2 M NaCl) | + | *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 | ||
+ | |||
+ | ===Sodium chloride - 5M 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 precipitation solution (with 1.2 M NaCl)=== | ||
+ | For 100 ml: | ||
+ | * 20 g PEG-8000 | ||
+ | * 7 g NaCl | ||
+ | * fill to 100ml with demiwater | ||
+ | Store at 4°C | ||
+ | |||
+ | [[Category:Schizophyllum]] |
Latest revision as of 17:15, 4 February 2021
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.
We were not as successful with this as hoped. We used the PEG-precipitation technique from this protocol and used it for a modified version of the DNA Extraction Phenol Chlorophorm S. commune protocol.
USE THIS PROTOCOL "DNA Extraction S. commune Phenol Chlorophorm PEG" FOR DNA EXTRACTIONS
- 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 4.5 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 to 65°С. This step cannot be omitted. Without heating up the CTAB/NaCl, the solution is sticky, and cannot be pipetted easily.
- 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.
- Incubate the tubes for 30 min at 4°C in the fridge.
- Allow the tubes to warm up to room temperature for ~3 min.
- 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.
- Separate the mixture into three phases by centrifuging at 10,000 xg for 15 min at 20°C.
- Transfer 4.5 ml of the upper aqueous phase to a fresh tube.
- Add 733 μL of 5M NaCl and mix by inversion. The final concentration NaCl should be 0.7 M.
- Add 594 μL of CTAB/NaCl solution (pH 8.0, preheated for >10min at step 4 ) and mix well by inverting multiple times.
- Incubate the tubes for 15 min at 65°C.
- Add 25 μL of DNase-free RNase A (100 mg/ml).
- Incubate the mixture for 30 min at 50°C.
- Add 5850 µl (an equal volume) of chloroform: isoamyl alcohol to the 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 a fresh tube.
- Add an equal volume of chloroform: isoamyl alcohol to the 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 a fresh tube.
- Add an equal volume of 20% PEG-8000, 1.2M NaCl and mix by inverting the tube multiple time.
- Collect the DNA by precipitation with 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 500 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 500 μL 70% ethanol.
- Centrifuge again at 10,000 xg for 1 min at room temperature.
- Remove the supernatant by aspiration
- Allow the pellet to dry in the air for 1–2 min.
- Elute the pellet in 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
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
Sodium chloride - 5M 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 precipitation solution (with 1.2 M NaCl)
For 100 ml:
- 20 g PEG-8000
- 7 g NaCl
- fill to 100ml with demiwater
Store at 4°C