Difference between revisions of "DNA Extraction Schizophyllum commune CTAB-PEG"
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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. | # 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 | + | # Add 163 μL of [[5M NaCl]] and mix by inversion. The final concentration NaCl should be 0.7 M. |
− | # Add 132 μL of CTAB/NaCl solution(pH 8.0, preheated at step 4) and mix well | + | # Add 132 μL of CTAB/NaCl solution (pH 8.0, preheated for >10min at step 4 ) and mix well by inverting multiple times. |
− | # 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. | + | # Incubate the tubes for 15 min at 65°C. |
+ | # 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. | # 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. | # 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. | ||
Line 50: | Line 52: | ||
===chloroform:isoamyl alcohol (24:1 v/v)=== | ===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 | ||
+ | '''Note that chloroform:isoamyl alcohol needs to be disposed of properly.''' | ||
===CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl)=== | ===CTAB/NaCl (10% w/v CTAB, 0.7 M NaCl)=== | ||
Line 64: | Line 69: | ||
* 20 g PEG-8000 | * 20 g PEG-8000 | ||
* 7 g NaCl | * 7 g NaCl | ||
+ | * fill to 100ml with demiwater |
Revision as of 16:41, 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 by inversion. The final concentration NaCl should be 0.7 M.
- Add 132 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.
- 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
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)
In a fume hood, mix in a 250 ml Greiner bottle:
- 96 ml Chloroform
- 4 ml Isoamyl alcohol
Note that chloroform:isoamyl alcohol 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
20% PEG 8000 solution (1.2 M NaCl)
For 100 ml:
- 20 g PEG-8000
- 7 g NaCl
- fill to 100ml with demiwater