实验概要

Plasmid DNA is isolated from large-scale (500 ml) bacterial cultures by treatment with alkali and SDS.

主要试剂

Buffers and Solutions

Alkaline lysis solution I

For preparations of plasmid DNA that are to be subjected to further purification by chromatography (please see

Chapter 1, Protocol 9 ), sterile Alkaline lysis solution I may be supplemented just before use with the appropriate

volume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 μg/ml

Alkaline lysis solution II

Alkaline lysis solution III

Antibiotic for plasmid selection

Chloramphenicol (34 mg/ml)

Ethanol

Isopropanol

STE

TE (pH 8.0)

Enzymes and Buffers

Lysozyme (10 mg/ml)

Restriction endonucleases

Media

Rich medium

实验步骤

1. Inoculate 30 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic either with a single

colony of transformed bacteria or with 0.1-1.0 ml of a small-scale liquid culture grown from a single colony.

2. Incubate the culture at the appropriate temperature with vigorous shaking until the bacteria reach late log phase (OD600

= approx. 0.6).

3. Inoculate 500 ml of LB, YT, or Terrific Broth medium (prewarmed to 37°C) containing the appropriate antibiotic in a 2-

liter flask with 25 ml of the late-log-phase culture. Incubate the culture for approx. 2.5 hours at 37°C with vigorous

shaking (300 cycles/minute on a rotary shaker).

4. For relaxed plasmids with low or moderate copy numbers, add 2.5 ml of 34 mg/ml chloramphenicol solution. The final

concentration of chloramphenicol in the culture should be 170 μg/ml.

For high-copy-number plasmids, do not add chloramphenicol.

5. Incubate the culture for a further 12-16 hours at 37°C with vigorous shaking (300 cycles/minute on a rotary shaker).

6. Remove an aliquot (1-2 ml) of the bacterial culture to a fresh microfuge tube and store it at 4°C. Harvest the remainder

of the bacterial cells from the 500-ml culture by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 15

minutes at 4°C. Discard the supernatant. Stand the open centrifuge bottle in an inverted position.

7. Resuspend the bacterial pellet in 200 ml of ice-cold STE. Collect the bacterial cells by centrifugation as described in

Step 6. Store the pellet of bacteria in the centrifuge bottle at -20°C.

8. Use one of the methods described in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 to prepare plasmid DNA from the 1-

2-ml aliquot of bacterial culture set aside in Step 6. Analyze the minipreparation plasmid DNA by digestion with the

appropriate restriction enzyme(s) and agarose gel electrophoresis to ensure that the correct plasmid has been

propagated in the large-scale culture.

9. Allow the frozen bacterial cell pellet from Step 7 to thaw at room temperature for 5-10 minutes. Resuspend the pellet in

18 ml (10 ml) of Alkaline lysis solution I.

10. Add 2 ml (1 ml) of a freshly prepared solution of 10 mg/ml lysozyme.

11. Add 40 ml (20 ml) of freshly prepared Alkaline lysis solution II. Close the top of the centrifuge bottle and mix the

contents thoroughly by gently inverting the bottle several times. Incubate the bottle for 5-10 minutes at room

temperature.

12. Add 20 ml (15 ml) of ice-cold Alkaline lysis solution III. Close the top of the centrifuge bottle and mix the contents gently

but well by swirling the bottle several times (there should no longer be two distinguishable liquid phases). Place the

bottle on ice for 10 minutes.

13. Centrifuge the bacterial lysate at 20,000g (11,000 rpm in a Sorvall GSA rotor) for 30 minutes at 4°C in a mediumspeed

centrifuge. Allow the rotor to stop without braking. At the end of the centrifugation step, decant the clear

supernatant into a graduated cylinder. Discard the pellet remaining in the centrifuge bottle.

14. Measure the volume of the supernatant. Transfer the supernatant together with 0.6 volume of isopropanol to a fresh

centrifuge bottle. Mix the contents well and store the bottle for 10 minutes at room temperature.

15. Recover the precipitated nucleic acids by centrifugation at 12,000g (8000 rpm in a Sorvall GSA rotor) for 15 minutes at

room temperature.

16. Decant the supernatant carefully, and invert the open bottle on a paper towel to allow the last drops of supernatant to

drain away. Rinse the pellet and the walls of the bottle with 70% ethanol at room temperature. Drain off the ethanol,

and use a Pasteur pipette attached to a vacuum line to remove any beads of liquid that adhere to the walls of the bottle.

Place the inverted, open bottle on a pad of paper towels for a few minutes at room temperature.

17. Dissolve the damp pellet of nucleic acid in 3 ml of TE (pH 8.0).

18. Purify the crude plasmid DNA either by column chromatography ( Chapter 1, Protocol 9 ), precipitation with

polyethylene glycol ( Chapter 1, Protocol 8 ), or equilibrium centrifugation in CsCl-ethidium bromide gradients ( Chapter

1, Protocol 10 and Chapter 1, Protocol 11 ).

19. Check the structure of the plasmid by restriction enzyme digestion followed by gel electrophoresis.