Method

1. Transfer 3 ml of the crude large-scale plasmid preparation to a 15-ml Corex tube and chill the solution to 0°C in an ice bath.

2. Add 3 ml of an ice-cold solution of 5 M LiCl to the crude plasmid preparation, mix well, and centrifuge the solution at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C.

3. Transfer the supernatant to a fresh 30-ml Corex tube. Add an equal volume of isopropanol. Mix well. Recover the precipitated nucleic acids by centrifugation at 12,000g (10,000 rpm in a Sorvall SS-34 rotor) for 10 minutes at room temperature.

4. Decant the supernatant carefully, and invert the open tube to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the tube with 70% ethanol at room temperature. Carefully discard the bulk of the ethanol, and then use a vacuum aspirator to remove any beads of liquid that adhere to the walls of the tube. Place the inverted, open tube on a pad of paper towels for a few minutes. The pellet should still be damp.

5. Dissolve the damp pellet of nucleic acid in 500 pl of TE (pH 8.0) containing RNase A. Transfer the solution to a microfuge tube and store it for 30 minutes at room temperature.

6. Extract the plasmid-RNase mixture once with phenol:chloroform and once with chloroform.

7. Recover the DNA by standard ethanol precipitation.

8. Dissolve the pellet of plasmid DNA in 1 ml of sterile H2O, and then add 0.5 ml of PEG-MgCl2 solution.

9. Store the solution for >10 minutes at room temperature, and then collect the precipitated plasmid DNA by centrifugation at maximum speed for 20 minutes at room temperature in a microfuge.

10. Remove traces of PEG by resuspending the pellet of nucleic acid in 0.5 ml of 70% ethanol. Collect the nucleic acid by centrifugation at maximum speed for 5 minutes in a microfuge.

11. Remove the ethanol by aspiration and repeat Step 10. Following the second rinse, store the open tube on the bench for 10-20 minutes to allow the ethanol to evaporate.

12. Dissolve the damp pellet in 500 pl of TE (pH 8.0). Measure the OD260 of a 1:100 dilution in TE (pH 8.0) of the solution, and calculate the concentration of the plasmid DNA assuming that 1 OD260 = 50 pg of plasmid DNA/ml.

13. Store the DNA in aliquots at -20°C.

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