Preparation
1. Take fresh (or frozen) calf thymus, remove blood and connective tissue, cut into small pieces on ice, weigh 20g, add 2 volumes of 0.1mol/L sodium chloride-0.05mol/L, pH=7.0 sodium citrate buffer solution, and homogenize at high speed for 5 minutes using a tissue homogenizer. 2. Centrifuge the tissue precipitate at 3000 rpm for 15 minutes. Wash the precipitate twice with 50 mL of the above buffer solution, homogenizing and washing with a homogenizer each time, centrifuging as before. 3. Add 6 volumes of 10% sodium chloride solution to the resulting nuclear precipitate, mix thoroughly, and refrigerate overnight to fully extract DNPs until the solution becomes viscous. 4. Slowly add the resulting translucent, viscous liquid to cold distilled water using a dropper, stirring gently while adding (final NaCl concentration is 0.14 mol/L). White filamentous material—nucleoprotein—will precipitate. Let the precipitate stand in the refrigerator for several hours and collect it. 5. Dissolve the precipitate again in four times its volume of 10% sodium chloride solution, stirring rapidly to accelerate dissolution. Add half a volume of chloroform-isoamyl alcohol mixture, shake vigorously for about 5 minutes, and centrifuge at 3000 rpm for 15 minutes to obtain three layers: the upper layer is an aqueous layer containing DNA and DNA nucleoproteins; the lower layer is an organic solvent layer of chloroform-isoamyl alcohol; and the denatured protein is between the two layers. 6. Aspirate the upper aqueous layer and then deproteinize with chloroform-isoamyl alcohol as before until no more denatured protein appears at the interface. 7. Aspirate the supernatant and inject it into twice the volume of 95% ethanol. Stir with a glass rod to precipitate the white fibrous DNA, drain, wash with 80% ethanol, and then wash with a small amount of anhydrous ethanol. After draining as much ethanol as possible, spread it on a petri dish. Dry in a vacuum desiccator to obtain white fibrous sodium DNA. DNA exists in organisms in the form of a complex with proteins; therefore, after extracting the deoxyribonucleic acid-protein complex (DNP), the proteins must be removed.
Uses
Deoxyribonucleic acid sodium salt, is exceptionally useful as a substrate for DNA polymerase assays, in the amplification of very long fragments and as a carrier DNA for precipitations. Calf thymus DNA is exceptionally useful as a substrate for DNA polymerase assays, in the amplification of very long fragments and as a carrier DNA for precipitations. It was used as a standard to determine DNA content in gels and for DNA quantification by fluorescent assay.
