PreparationofPlasmidDNAbyAlkalineLysiswithSDS:Minipreparation

實驗概要

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS.

主要試劑

Buffers and Solutions:

    Alkaline lysis solution I

    Alkaline lysis solution II

    Alkaline lysis solution III

    Antibiotic for plasmid selection

    Ethanol

    Phenol:chloroform (1:1, v/v)

    STE

    TE (pH 8.0) containing 20 μg/ml RNase A
 

Media:

    Rich medium

實驗步驟

1. Inoculate 2 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. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge.Store the unused portion of the original culture at 4°C.

3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.

4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.

5. Add 200 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 150 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 the supernatant to a fresh tube.

8. (Optional) 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 2 volumes of ethanol 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 4°C 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. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.

12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.

13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube.

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 (5-10 minutes).

15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C.