Isolation and purification of animal genomic DNA
Isolation and purification of animal genomic DNA
The purpose of this experiment is to master the basic principles and methods of mass preparation of animal genomic DNA by salt solubilization method, according to the different solubility of ribonucleoprotein and deoxyribonucleoprotein in a certain concentration of sodium chloride solution for separation, and then remove the protein with protein denaturing precipitant, so that the nucleic acid is released, and then use the nature of nucleic acid insoluble in ethanol nucleic acid precipitation, to achieve the purpose of separation and purification.
Operation method
salting out
Principle
According to the different solubility of ribonucleoprotein and deoxyribonucleoprotein in a certain concentration of sodium chloride solution to separate, and then use protein denaturing precipitant to remove protein, so that nucleic acid release, and then use the nature of nucleic acid insoluble in ethanol nucleic acid precipitation, to achieve the purpose of separation and purification. In 0.14 mol/ L sodium chloride, the nucleic acid was dissolved in ethanol. 14 mol / L of sodium chloride solution, RNA nucleoprotein (RNP) solubility, while the DNA nucleoprotein (DNP) solubility is small; on the contrary, in 1 mol / L of sodium chloride solution, DNP solubility is the largest, while RNP solubility is very small, so that the DNA, RNA nucleoproteins separate. After the separation of nuclear proteins can be used to protein denaturing precipitant (chloroform + isoamyl alcohol, sodium dodecyl sulfate, hot phenol, etc.) to remove the protein, the release of nucleic acid, nucleic acid will be precipitated from the solution. Animal liver contains ribonuclease (RNase) and deoxyribonuclease (DNase), so it is necessary to maintain a low temperature, to prevent the activation of ions such as Mg2+, Fe2+ and Co2+.
Materials and Instruments
Protein Move Fresh pig liver 4 g, washed with SC solution to remove blood, cut at low temperature, add 8 ml of the above solution, and continue to mash. The homogenate was centrifuged at 4000 r/min for 10 min, the upper layer was RNP extract, and the lower layer was DNP and cell debris. The homogenate was centrifuged at 4000 r/min for 10 min, the upper layer was RNP extract, and the lower layer was DNP and cell debris. The upper layer is RNP extract, the lower layer is DNP and cell debris. RNA preparation), and the lower layer was extracted twice with 5 ml of SC solution to minimize the impact of RNP on DNP extraction. The lower layer was then extracted twice with 5 ml of SC solution to minimize the effect of RNP on DNP extraction. The lower layer of precipitate was transferred to a triangular flask, add 20 ml of the same solution, mix well, add 4 ml of 5% SDS and mix well. 4 ml 5% SDS, add 15 ml chloroform/isoamyl alcohol ( 20/ 1 ) mixture, add solid sodium chloride while shaking to reach a final concentration of 1 mol/ L, and shaking for 30 min. Centrifuge at 4000 r/min for 20 min, carefully remove the tube and observe three layers, the upper layer is the aqueous phase (the DNA is dissolved here) and the upper layer is the aqueous phase (the DNA is dissolved here). The upper layer is aqueous phase (DNA is dissolved here), the middle layer is milky white protein precipitation layer, and the lower layer is chloroform layer. The upper layer is aqueous phase (DNA is dissolved here), the middle cream color is protein precipitation layer, and the lower layer is chloroform layer. Pipette the upper layer carefully. Remove the protein in the same way until there is no protein precipitate in the middle layer. The protein is removed in the same way until there is no protein precipitate in the middle layer. After measuring the volume of the upper aqueous phase, add an equal volume of cold ethanol (95%) to the beaker. Stirring (in one direction) while adding, the glass rod is wrapped with fibrous DNA, when all the DNA is wrapped, squeeze the glass rod. When all the DNA is wound, squeeze it dry, wash it again with anhydrous ethanol, and remove it to a desiccator for drying. Remove and dry in a desiccator. Weigh and calculate the yield. For more product details, please visit Aladdin Scientific website.
Sodium citrate Sodium chloride Distilled water Hydrochloric acid SDS Ethanol Chloroform Isoamyl alcohol
Freezing centrifuge Tissue masher Beaker Measuring cylinder Glass rod Triangular flask Centrifuge tube