Removal of small fragments of nucleic acids from plasmid DNA preparations by lithium chloride precipitation
Removal of small fragments of nucleic acids from plasmid DNA preparations by lithium chloride precipitation
The principle of lithium chloride precipitation for removing small fragments of nucleic acids (both DNA and RNA) from plasmid preparations is based on the fact that the two types of nucleic acids differ in their solubility in lithium chloride solution. Lithium chloride is a strong dehydrating agent that reduces the solubility of RNA ( Hearst and Vinograd 1961a, b) and strips proteins from chromatin (Kondo 1979). As a result, high molecular mass RNA and proteins in plasmid extracts can form precipitates in highly concentrated lithium chloride solutions, which can be separated by low-speed centrifugation (for an example, see Kondo et al. 1991). This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Removal of small fragments of nucleic acids from plasmid DNA preparations by lithium chloride precipitation
Principle
The principle of lithium chloride precipitation for removing small fragments of nucleic acids (both DNA and RNA) from plasmid preparations is based on the fact that the two types of nucleic acids differ in their solubility in lithium chloride solution. Lithium chloride is a strong dehydrating agent that reduces the solubility of RNA ( Hearst and Vinograd 1961a, b) and strips proteins from chromatin (Kondo 1979). As a result, high molecular mass RNA and proteins in crude plasmid extracts can form precipitates in highly concentrated lithium chloride solutions, which can be separated by low-speed centrifugation (for an example, see Kondo et al. 1991).
Materials and Instruments
DNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ethanol Isopropanol LiCl Sodium acetate TE
Sorvsll SS-34 rotor or equivalent replacement rotor
1. Buffers and solvents
Ethanol, isopropanol, LiCl ( 4 mol/L), sodium acetate (3 mol/L, pH 5.2), TE ( pH 8.0) containing RNase A at a concentration of 20 μg/ml RNase A ( pH 8.0).
2. Nucleic acids and oligonucleotides
DNA samples were purified by density gradient centrifugation with CsCl
3. centrifuge and rotor
Sorvsll SS-34 rotor or alternative rotor of equal performance
II. Methods
1. Determine the volume of the plasmid preparation by adding 0.1 v/v of 3 mol/L sodium acetate (pH 5.2) and 2 times the volume of ethanol, and leave the mixture at 4 °C for 30 min.
2. Recover the nucleic acid precipitate by centrifugation at >10,000 g (>9000 r/min, using Sorvsll SS-34 rotor) for 15 min at 4°C, and decant as much of the supernatant as possible. The tubes were then uncapped and placed on a bench for several minutes to evaporate the ethanol.
3. Dissolve the wet nucleic acid precipitate with 1 ml of TE (pH 8.0) containing RNase A (concentration ≥ 100 μg/ml).
4. Add 3 ml of 4 mol/L LiCl solution and place on ice for 30 min.
5. Centrifuge at 12000 g (10000 r/min with Sorvsll SS-34 rotor) for 15 min at 4°C to isolate the DNA plasmid from the precipitated nucleic acid.
6. Transfer the supernatant to a new centrifuge tube, add 6 ml of isopropanol and leave for 30 min at room temperature to precipitate the plasmid DNA.
7. Centrifuge at 12000 g (10000 r/min, using Sorvsll SS-34 rotor) at 4℃ for 15 min to recover the precipitated plasmid DNA.
8. Carefully decant the supernatant, add 5-10 ml of 70% ethanol, briefly shake the centrifuge tube, and centrifuge at 12000 g for 10 min at 4℃.
9. Carefully decant the supernatant, uncap the tube and place it on the bench for a few minutes until the ethanol evaporates.
10. Dissolve the wet DNA precipitate with TE (pH 8.0).