Extract DNA from animal liver

First, the principle:

In concentrated sodium chloride (1-2 mol/L) solution, the solubility of deoxyribonucleoprotein is very large, and the solubility of ribonucleoprotein is very small. In dilute sodium chloride (0.14 mol/L) solution, deoxyribonucleoprotein The solubility is small and the solubility of ribonucleoprotein is large. Therefore, deoxyribonucleoprotein and ribonucleoprotein can be extracted from the sample separately using different concentrations of sodium chloride solution.

The extracted nuclear protein is treated with SDS (sodium dodecyl sulfate), and the DNA or (RNA) is separated from the protein, and the protein precipitate can be removed by chloroform-isoamyl alcohol, and the DNA is dissolved in the solution. When an appropriate amount of ethanol is added to the solution, the DNA is precipitated.

In order to prevent DNA or (RNA) enzymatic hydrolysis, EDTA (ethylenediaminetetraacetic acid) is added during extraction.

Second, experimental materials and instruments:

1, the mouse liver

2, homogenizer

3, centrifuge 5000r / min

4. Measuring cylinder 50ml (×1), 25 ml (×1), 10 ml (×1)

5, straw 5 ml (×2)

6, water bath

7, vacuum dryer

Third, the reagent:

1.5 mol/L NaCl solution: 292.3 g of NaCl was dissolved in water and diluted to 1000 ml.

2.0.14 mol/L NaCl - 0.15 mol/L EDTA-Na solution: Dissolve 8.18 g of NaCL and 37.2 g of EDTA-Na in distilled water and dilute to 1000 ml.

3.25% SDS solution: dissolve 25g sodium lauryl sulfate in 100ml 45% ethanol

4.0.015 mol/L NaCL-0.0015 mol/L Trisodium citrate solution: 0.828 g of sodium chloride and 0.341 g of trisodium citrate are dissolved in distilled water and diluted to 1000 ml.

5. Chloroform-isoamyl alcohol mixture: chloroform: isoamyl alcohol = 24:1 (V/V).

6.1.5 mol/L NaCL-0.15 mol/L trisodium citrate solution: sodium chloride 82.8 g and trisodium citrate 34.1 g were dissolved in distilled water and diluted to 1000 ml.

7.3 mol/L sodium ethoxide-0.001 mol/L EDTA-Na solution: Weigh 408 g of sodium acetate, and EDTA-Na 0.372 g is dissolved in distilled water and diluted to 1000 ml.

8.70% ethanol, 80% ethanol, 95% ethanol, absolute ethanol.

9. Diphenylamine reagent

10.1 mol/L perchloric acid solution: 10 ml of perchloric acid (70%) was diluted to 110 ml with distilled water.

Fourth, the operation:

1. Isolation and purification of DNA:

(1) 10 mice were quickly killed, the liver was removed, and the plasma was washed away with 0.14 mol/L NaCl-0.15 mol/l EDTA solution, and cut into 50 ml of 0.14 mol/L NaCl-0.15 mol/l EDTA solution. Grinding in the slurry, after grinding into a paste, the paste is centrifuged for 10 minutes (4000r/min), the supernatant is discarded, and the precipitate is washed two or three times with 0.14mol/L NaCl-0.15mol/L EDTA solution. The precipitate is a crude deoxyribonucleoprotein.

(2) Add 0.14mol/L NaCl-0.15mol/L EDTA solution to the above precipitate to make the total volume of 44ml, then add 3ml of 25% SDS solution, stir while adding, add 60 water bath for 10 minutes. The solution was viscous and slightly transparent, and was taken out to cool to room temperature. This step is to separate the nucleic acid from the protein.

(3) Add 10 ml of 5 mol/L NaCL to make the final concentration of NaCl reach 1 mol/L, stir for 10 minutes, add about one volume of chloroform-isoamyl alcohol mixed solution for about 20 minutes, shake for 20 minutes, and centrifuge for 10 minutes (4000 r/min). The precipitate was removed, and the supernatant was slowly added 1.5 to 2 times 95% ethanol. The DNA precipitate was precipitated and slowly stirred with a glass rod, and the DNA filament was wrapped around the glass rod.

(4) Put the crude DNA in 27ml 0.015 mol / L NaCl -0.0015 mol / L trisodium citrate solution, then add 3ml 1.5 mol / L NaCl -0.15 mol / L trisodium citrate solution, stir well, add One volume of chloroform-isoamyl alcohol mixture was shaken for ten minutes, centrifuged (4000 r/min, 10 minutes), the upper liquid was decanted (precipitated and discarded), and 1.5 volumes of 95% ethanol was added thereto, and the DNA was precipitated. After centrifugation, the supernatant was discarded and the pellet (crude DNA) was processed once in this procedure.

(5) Precipitating the above step in 27ml 0.015 mol/L NaCl-0.0015 mol/L trisodium citrate solution, then adding 2 times 95% ethanol in a linear form, stirring and removing the filamentous DNA, followed by Wash once with 70%, 80%, 95% and absolute ethanol and dry in vacuo.

2. Identification: DNA was determined by the diphenylamine method.

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