ã€Fundamental】
The DNA of eukaryotes mainly exists in the nucleus and exists as a double-stranded linear polymer. The main process of DNA extraction is to break up tissues and cells, remove proteins and other macromolecules such as polysaccharides and lipids. This experiment denatures proteins by proteinase K and SDS, and then removes proteins with phenol/chloroform. The DNA was precipitated, and the DNA content was detected by A260/A280, and then identified by agarose gel electrophoresis.
ã€equipment】
Glass homogenizer
2. Benchtop centrifuge
ã€Reagents】
1. DNA extraction buffer:
10mmol/L Tris-Cl (pH 8.0)
0.1mol/L EDTA (pH 8.0)
20μg/ml pancreatic RNase
0.5% SDS
2. Proteinase K 20mg/ml
3. Saturated phenol
4.NaAC (pH 5.2) 3 mol / L
5.1×TE (pH 8.0)
6.100% ethanol
7.70% ethanol
ã€Steps】
1. Take fresh liver tissue 0.2g, add 1.2ml DNA extraction buffer to make homogenate, do not break the nucleus.
2. Add 6 μl RNase (20 mg/ml), pour into Ep tube, incubate in 37 °C water bath for 30 min, then add 6 μl proteinase K (final concentration 50 μg/ml), and incubate at 50 °C for 2 h.
3. Take 0.5ml of heat preservation solution, add 0.5ml of saturated phenol, mix well by shaking, centrifuge at 10 000rpm for 10 minutes, transfer the upper aqueous phase to Ep tube, add equal volume of chloroform / isoamyl alcohol (24:1). After mixing well, centrifuge at 10 000 rpm for 10 min.
4. Transfer the upper aqueous phase to the Ep tube, add 50 μl of 3 mol/L NaAc, add 2-2.5 volumes of absolute ethanol, and centrifuge as above.
5. Carefully discard the supernatant, centrifuge and add 70% ethanol to wash once, retain the precipitate, let it dry naturally, add appropriate amount of water or TE to dissolve.
6. Perform nucleic acid quantitative determination and electrophoresis identification.
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