DNA extraction is mainly CTAB method, and other methods include physical methods such as glass bead method, ultrasonic method, grinding method, and freeze-thaw method. Chemical methods such as guanidinium isothiocyanate method, alkaline lysis method. Biological mode: enzymatic method. Depending on the method of nucleic acid separation and purification; siliceous materials, anion exchange resins, and the like.
method one)
Reagents:
1. Extraction buffer, 2% CTAB (W/V), 2% PVP K25 (w/v) (de-pigmentation), 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, (pH 8.0), 2.0 M NaCl. Shanghai Chuangsai provides ethylenediaminetetraacetic acid (EDTA) standard solution, 60-00-4, commodity number: D16-1014183-concentration: 0.1N/L, price 288 yuan.
2.10M LiCl
3. 2M LiCl
4.DEPC-water (inhibition of RNase activity)
5. 3M NaAc (pH 5.2)
6. 96% ethanol
7. 70% ethanol
step:
1. The extraction buffer is preheated at 65 ° C;
2. Add the bacteria to the pre-warmed buffer (700 ul), 65 ° C, 10 min;
3. Add phenol/chloroform/isoamyl alcohol (25:24:1), shake, centrifuge 12,000 rpm, 10 min;
4. Take the supernatant, add chloroform / isoamyl alcohol (24:1), shake, centrifuge 12,000 rpm, 10min;
5. Take the supernatant and repeat 4 steps;
6. Add 1/10 volume of 3M sodium acetate and 1.5 volumes of ethanol (or add an equal volume of isopropanol), -20C precipitation;
11. Centrifuge at 12,000 rpm for 10 min;
12. Discard the supernatant, wash with 70% ethanol, or wash with 100% ethanol, then dissolve in 100 ml of water.
Method Two)
1. The strain was inoculated into liquid LB medium and incubated overnight at 37 °C with shaking.
2. Centrifuge 1.5 ml of culture at 12000 rpm for 2 min.
3. The pellet was added to 567 ul of TE buffer, repeatedly boiled and resuspended, 30 ul of 10% SDS and 15 ul of proteinase K were added, mixed, and incubated at 37 ° C for 1 h. Shanghai Chuangsai provides sodium dodecyl sulfate (SDS), AR, 92.5-100.5%, 151-21-3, commodity number: C16-S10436-500g, price 50 yuan.
4. Add 100ul of 5mol/L NaCl, mix well, add 80ul CTAB/NaCl solution, mix and incubate for 10min at 65°C.
5. Add an equal volume of phenol/chloroform/isoamyl alcohol, mix and centrifuge for 4-5 minutes, transfer the supernatant to a new tube, add 0.6-0.8 volumes of isopropanol, and gently mix until the DNA is precipitated. The precipitate can be centrifuged slightly. Shanghai Chuangsai provides isopropyl alcohol, 99.5%, ultra-dry, containing molecular sieve, water ≤ 50ppm, 67-63-0, commodity number: C52-H11026-100ml, price 150 yuan.
6. After the precipitate was washed with 1 ml of 70% ethanol, the ethanol was discarded by centrifugation.
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