After the initial characterization, it is possible to purify further some or all of the plasmid DNAs by RNase digestion and extraction with organic solvents. This further purified DNA is suitable for techniques such as DNA sequencing, subcloning or the production of gene probes. In order to purify plasmid DNA after the isolation process, any residual RNA and contaminating protein are removed. This purification step involves two main steps, which are, first, removing residual RNA by using RNase in order to digest RNA and, second, extract contaminating protein using organic solvents, phenol-chloroform.
Materials:
- RNase A: Make up as a solution in water at 10 mg/mL, Heat for 10 min in a boiling water bath or heating block to eliminate any DNase activity. Aliquot and store at -20oC.
- 0.4 M Ammonium acetate.
- Chloroform= A 24: 1 mix of chloroform and isoamyl alcohol. Store at 4oC.
- Phenol/chloroform= 25:24: 1 mix of TE-equilibrated phenol, chloroform, and isoamyl alcohol. Store at 4oC.
- 100% Ethanol.
- Sterile wooden toothpicks.
Methods:
- Add 50 microliters of 4 M ammonium acetate containing 200 micrograms/mL RNase A to each miniprep and incubate it at room temperature for 20 min.
- Add 100 microliters of phenol/chloroform to each DNA preparation.
- Vortex briefly and centrifuge at high speed for 2 min in a microfuge. Remove the top layer containing the DNA and place it in a new sterile tube.
- Add 100 microliters of chloroform to each tube.
- Vortex briefly and centrifuge at high speed in a microfuge for 2 min. Remove the DNA in the top layer and place it in a second sterile tube.
For phenol/chloroform extractions avoid removing material from the interface.
- Add 200 microliters of 100% ethanol to each tube.
- Shake briefly to precipitate the DNA and centrifuge at high speed for 5 min at room temperature.
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