Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation
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实验试剂
For preparations of plasmid DNA that are to be subjected to further purification by chromatography , sterile Alkaline lysis solution I may be supplemented just before use with the appropriatevolume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 μg/ml.
实验步骤
1. Inoculate 10 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.
2. Transfer the culture into a 15-ml tube and recover the bacteria by centrifugation at 2000g (4000 rpm in a Sorvall SS-34rotor) for 10 minutes at 4°C.
4. Resuspend the bacterial pellet in 200 μl of ice-cold Alkaline lysis solution I by vigorous vortexing, and transfer the suspension to a microfuge tube.
5. Add 400 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice.
6. Add 300 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.
7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer 600 μl of the supernatant to a fresh tube.
8. Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.
9. Precipitate nucleic acids from the supernatant by adding 600 μl of isopropanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.
10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at room temperature in a microfuge.
11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Remove any drops of fluid adhering to the walls of the tube.
12. Add 1 ml of 70% ethanol to the pellet and recover the DNA by centrifugation at maximum speed for 2 minutes at room temperature in a microfuge.
14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (2-5 minutes).
15. Dissolve the nucleic acids in 100 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase).Vortex the solution gently for a few seconds and store at -20°C.
