1. Take 100ul-200ul of fresh or frozen muscle tissue or 0.05g of sample tissue preserved in 95% ALC. 2. Cut the tissue as much as possible and put them into 1.5ml centrifuge tubes.
3. Add 450ul of lysis buffer (10mM Tris-HCl pH8.0; 100mM EDTA, pH8.0) to each tube
4. Add 10% SDS 50-100ul (70-80 variable) (1-2%) to each tube, then add 2.5-5ul (3ul) proteinase K (20mg/ml) to make the final concentration 100ug/ml. Mix well, 55°C for 3h to overnight (invert the tube several times during this period).
5. Add 150ul NaCl (salting out) to the sample, centrifuge at 8000 rpm at room temperature for 20 minutes to remove precipitation, and then add the supernatant
Add an equal volume (500ul) of pre-cooled isopropanol (precipitated DNA), mix well, treat at 20°C for 30 minutes, centrifuge at 14000 rpm for 10 minutes, and remove the supernatant. The precipitate is washed with 70% ALC and centrifuged to remove ALC. Volatilize ALC at room temperature for 5-10 minutes. Add 500ul TE and 10ul RNAase (final concentration 4mg/ul), which is 2.5ul. 37℃ 30min or 65℃ 15min.
6. Add an equal volume of acid solution (to extract protein from DNA), slowly invert the centrifuge tube back and forth for 10 minutes, and centrifuge at 13000-15000 rpm for 10 minutes. Use a pipette (large tip tip) to carefully remove the upper phase to another clean centrifuge tube, do not touch the white protein layer between the two phases (repeat the operation once as appropriate, using dna extractor, it can be extracted automatically , faster and more efficient.).
7. Add an equal volume of phenol: chloroform (reduced phenol) (1:1), shake gently, slowly mix by inversion for 10 minutes, centrifuge at 13000-15000 rpm for 5-10 minutes, and take the supernatant in a clean centrifuge tube.
8. Add equal volume of chloroform (chloroform: isoamyl alcohol (to prevent chloroform volatilization) = 24:1), shake gently, and slowly invert and mix well
Centrifuge at 13000-15000rpm for 5-10min for 10min. Take the supernatant and add 1/10 volume of 2M NaCl and equal volume of absolute ethanol (or equal volume of isopropanol) stored at -20°C (precipitate DNA), precipitate the DNA for 10 min, centrifuge at 13000 rpm for 5 min, remove the supernatant, and add 100- 150ul 70% ALC, centrifuge, discard ALC carefully (beware of loss of DNA precipitation), wash again with 70% ALC, and then remove ALC. Dry in an oven at 45°C for 15-30min, rinse with double distilled water along the pipe wall (the amount of TE depends on the amount of DNA precipitation, between 80-250ul), dissolve for 12-24h, store at -20°C for later use.
2. Matters needing attention:
1. The lysate should be preheated to inhibit DNase, accelerate protein denaturation, and promote DNA dissolution.
2. Phenol must be alkali-balanced. Phenol is highly corrosive, splashing on the skin, mucous membranes and eyes can cause damage, so care should be taken to protect it. Chloroform is flammable, explosive, volatile, and has a neurotoxic effect. Pay attention to protection during operation.
3. The operation steps should be gentle and minimize the artificial degradation of DNA as much as possible.
4. When taking each supernatant, it should not be too greedy to prevent interference from non-nucleic acid components.
5. Isopropanol, ethanol, NaAc, KAc, etc. should be pre-cooled to reduce the degradation of DNA, promote the phase separation of DNA and protein, and DNA precipitation.
6. The reagents and equipment used in the process of DNA extraction should be processed without nuclease by high-pressure baking and other methods.
7. All reagents are prepared with autoclaved double distilled water