Animal DNA Extraction

Summary

Animal liver DNA extraction can be (1) used for forensic identification, (2) for diagnostic and phylogenetic studies, and (3) for further polymerase chain reaction (PCR) to obtain DNA.

Operation method

concentrated salting

Principle

In concentrated sodium chloride (1-2 mol/L) solution, the solubility of deoxyribonucleoproteins is large and that of ribonucleoproteins is small, and in dilute sodium chloride (0.14 mol/L) solution, the solubility of deoxyribonucleoproteins is small and that of ribonucleoproteins is large. Therefore, deoxyribonucleoproteins and ribonucleoproteins can be extracted separately from the sample using different concentrations of sodium chloride solutions.

Materials and Instruments

Mouse liver
NaCl EDTA-Na SDS Trisodium citrate Chloroform-isoamyl alcohol mixture Ethanol Diphenylamine Anhydrous ethanol Perchloric acid
Homogenizer Centrifuge Measuring cylinder Straws Water bath Vacuum drier

Move

I. Reagent Preparation
1. 5 mol/L NaCl solution: Dissolve 292.3 g NaCl in water and dilute to 1 000 ml.
2. 0.14mol/L NaCl-0.15mol/L EDTA-Na solution: Dissolve 8.18g NaCL and 37.2g EDTA-Na in distilled water and dilute to 1 000 ml.
3. 25% SDS solution: Dissolve 25 g of sodium dodecyl sulfate in 100 ml of 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 1 000 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: 82.8 g of sodium chloride and 34.1 g of trisodium citrate are dissolved in distilled water and diluted to 1,000 ml.
7. 3 mol/L sodium ethanol 0.001 mol/L EDTA-Na solution: Weigh 408 g of sodium acetate and 0.372 g of EDTA-Na, dissolve in distilled water and dilute to 1 000 ml.
8. 70% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol.9. 1 mol/L perchloric acid solution: Dilute 10 ml of perchloric acid (70%) to 110 ml with distilled water.
II. Isolation and purification of DNA
1. 10 mice were killed quickly, the liver was removed, washed with 0.14 mol/L NaCl 0.15 mol/l EDTA solution to remove plasma, cut, add 50 ml of 0.14 mol/L NaCl -0.15 mol/L EDTA solution, placed in a homogenizer to grind, to be ground to a paste, centrifugation of the paste for 10 minutes (4,000 r/min), and the precipitate was washed two or three times with 0.14 mol/L NaCl-0.15 mol/L EDTA solution, the resulting precipitate was deoxyribonucleoprotein crude product. The supernatant was discarded, and the precipitate was washed two or three times with 0.14 mol/L NaCl-0.15 mol/L EDTA solution, and the resulting precipitate was the crude deoxyribonucleoprotein.
2. Add 0.14 mol/L NaCl-0.15 mol/L EDTA solution to the above precipitate to make the total volume of 44 ml, then add 25% SDS solution 3 ml dropwise, stirring while adding, after adding, put the 60 water bath and keep warm for 10 minutes (non-stop stirring), the solution becomes viscous and slightly transparent, take out and cool down to room temperature. This step is to separate nucleic acid from protein.
3. Add 5 mol/L NaCl 10 ml, so that the final concentration of NaCl reaches 1 mol/L, stirring for 10 minutes, add about double the volume of chloroform-isoamyl alcohol mixture, shaking for 20 minutes, centrifugation for 10 minutes (4,000 r/min). The precipitate was removed, and 1.5-2 times 95% ethanol was added to the upper layer of the clear liquid, and the DNA precipitate was precipitated, and the DNA filaments were entangled in the glass rod when it was stirred slowly with a glass rod.
4. Put the crude DNA in 27 ml 0.015 mol/L NaCl-0.0015 mol/L trisodium citrate solution, then add 3 ml 1.5 mol/L NaCl-0.15 mol/L trisodium citrate solution, stir well, add double the volume of chloroform-isoamyl alcohol mixture, shaking for ten minutes, centrifugation (4,000 r/min, 10 minutes), pour out the upper layer of liquid (precipitate discarded), add 1.5 times the volume of 95% ethanol. /min, 10 minutes), decant the upper layer of liquid (precipitate discarded), add 1.5 times the volume of 95% ethanol, DNA precipitation precipitation precipitation. Centrifuge, discard the supernatant, and repeat this procedure once for the precipitate (crude DNA).
5. The precipitate obtained in the previous step was placed in 27 ml of 0.015 mol/L NaCl-0.0015 mol/L trisodium citrate solution, and then 2 times of 95% ethanol was added in a linear fashion, stirring as it was added, and the filamentous DNA was taken out, washed with 70%, 80%, 95%, and anhydrous ethanol once each, and dried in a vacuum.

Caveat

To prevent DNA or (RNA) digestion, EDTA (ethylenediaminetetraacetic acid) is added during extraction.

Common Problems

Reagent preparation

1. 5 mol/L NaCl solution: Dissolve 292.3 g NaCl in water and dilute to 1 000 ml.

2. 0.14mol/L NaCl-0.15mol/L EDTA-Na solution: dissolve 8.18g NaCL and 37.2g EDTA-Na in distilled water, dilute to 1 000 ml. 3. 25% SDS solution: dissolve 25 g of dodecyl dodecyl sulfate (DDS) in water, dilute to 1 000 ml.

3. 25% SDS solution: Dissolve 25 g of sodium dodecyl sulfate in 100 ml of 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 dissolved in distilled water and diluted to 1 000 ml. 5.

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: 82.8 g of sodium chloride and 34.1 g of trisodium citrate were dissolved in distilled water and diluted to 1 000 ml. 7. 3 mol/L ethanol was dissolved in distilled water.

7. 3 mol/L sodium ethanol 0.001 mol/L EDTA-Na solution: weighing sodium acetate 408 g, EDTA-Na 0.372 g dissolved in distilled water, diluted to 1 000 ml.

8. 70% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol.

9. 1 mol/L perchloric acid solution: 10 ml of perchloric acid (70%) was diluted to 110 ml with distilled water.

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