Molecular Biology. Practical Guide - Velikov V.A. 2013

Isolation of Plasmid and Phage DNA
Mini-preparation of Plasmid DNA (Miniprep)

A plasmid is an extrachromosomal, self-replicating genetic element (a hereditary factor) found in Bacteria and certain other organisms, notably Yeasts. All DNA Cloning vectors have been derived from either Plasmids or Viruses. Vectors form the foundation of both genetic and Protein Engineering. In addition, recombinant DNA—that is, vector DNA combined with a DNA sequence of interest to the researcher—is widely used in Cell engineering, enzymology engineering, Gene Therapy, and other fields.

A plasmid typically exists as a circular (very rarely linear) double-stranded DNA molecule capable of autonomous Replication within a host cell. A vector plasmid serves as a DNA carrier.

Among the various Methods for isolating plasmid DNA, modifications of the alkaline lysis technique developed by Birnboim and Doly in 1979 (Birnboim, Doly, 1979) are the most widely used. These methods allow for the efficient Separation of plasmid DNA from high-molecular-weight chromosomal DNA. The peptidoglycan (murein) Cell wall is digested with Lysozyme, and the host cell membrane is dissolved using the detergent SDS under alkaline conditions at approximately pH 12.5. Under these conditions, the chromosomal DNA undergoes irreversible fragmentation and Denaturation. Plasmid DNA also denatures, but due to its small size, it does not fragment, and the two DNA strands remain intertwined. Upon lowering the pH by adding a potassium acetate solution (pH 5.0), the two strands of the plasmid renature with each other. Single-stranded chromosomal fragments from different Cells randomly reassociate (rehybridize) to form a high-molecular-weight, curd-like precipitate. This precipitate is easily removed by centrifugation, leaving the plasmid in solution, which is then recovered by ethanol precipitation. Because DNA in vivo is naturally bound to Proteins, a subsequent deproteinization step is required.

The replicative form of phages, which exists inside infected bacterial cells, also consists of double-stranded circular DNA molecules and can be readily isolated using the alkaline lysis method.

The protocol presented here is a Modification of the alkaline lysis method for bacterial cells, recognized as one of the best Procedures for obtaining high-quality plasmid DNA. Its main advantages are simplicity and high speed, comparable only to spin-Column DNA purification. The yield of DNA is sufficient for multiple restriction digests. For large-scale projects, the maxiprep Procedure is employed (Exercise 4.2).

The pBR322 plasmid, which serves as the source of DNA in this procedure, is one of the earliest molecular cloning vectors (Bolivar et al., 1977).

Materials and Equipment

E. coli strain XL1-Blue (Stratagene^TM) transformed with plasmid pBR322 (see App. 1, 4), microcentrifuge, orbital shaker.

Solutions

- Solution I for plasmid DNA isolation: 50 mM glucose; 100 mM Tris-HCl, pH 8.0; 10 mM EDTA. Store at +4°C; add lysozyme to a final concentration of 5 mg/ml just before use.

- Solution II ^а: 0.2 N NaOH; 1% SDS. Prepare fresh before use; brief storage at room Temperature in tightly closed plastic containers is permissible.

- Solution III: 3 M potassium acetate, pH 5.0. For 100 ml: 5 M potassium acetate — 60 ml; glacial CH₃COOH — 11.5 ml; H₂O — 28.5 ml.

- Phenol-chloroform mixture (Exercise 1.1)

- Chloroform-isoamyl alcohol mixture (Exercise 1.1).

- Ampicillin. Solution at 100 mg/ml in Water.

Procedure

1. Grow an E. coli cell culture containing plasmid pBR322 in 5 ml of 2YT nutrient medium (Topic 1) supplemented with 100 µg/ml of the antibiotic ampicillin for 16–24 hours on a thermostated rotary shaker at 37°C and 200–300 rpm.

2. Pellet the cells from the culture medium. To do this, transfer 1.5 ml of the E. coli overnight culture into a 1.7-ml microcentrifuge tube and centrifuge for 2 min at 10,000 rpm. Discard the supernatant by decanting. Repeat The Cell collection step in the same tube two more times.

3. Centrifuge for an additional 10 s and remove any residual culture medium using a micropipette.

4. Resuspend the pellet in 200 µl of Solution I using a micropipette or vortex mixer. Leave at room temperature for 5 min ^б.

5. Add 400 µl of Solution II, immediately mix vigorously by inverting the tube 5 times. This causes bacterial lysis and alkaline denaturation of the DNA. Place the samples on ice (0°C) for 5 min ^в.

6. Add 300 µl of ice-cold Solution III, invert the tube 5 times, and incubate on ice for 5 min.

7. Centrifuge for 5 min at maximum centrifuge speed to pellet the chromosomal DNA.

8. Using a micropipette, transfer the supernatant containing the plasmid DNA into a new tube containing 500 µl of isopropanol, mix by inversion, and let stand at room temperature for 10 min.

9. Pellet the plasmid by centrifugation for 10 min at maximum speed, pour off the supernatant, and remove any remaining liquid with a micropipette after a brief pulse centrifugation.

10. Dissolve the plasmid pellet in 100 µL of TE buffer (200 µL may also be used).

11. The plasmid DNA solution must be further purified by phenol deproteinization of the sample. To do this, add an equal volume of phenol-chloroform mixture to the tube, mix thoroughly, and separate the phases by centrifugation (10 min, 10,000 rpm).

12. Transfer the upper aqueous phase to a new tube, add an equal volume of a chloroform-isoamyl alcohol mixture (this not only denatures proteins but also removes residual phenol), mix thoroughly, and separate the organic and aqueous phases by centrifugation for 3 min.

13. Collect the upper aqueous phase, add 1/10 volume of cold 3M potassium acetate solution, pH 5.0, and precipitate the DNA with ethanol. To do this, add 1 mL of cold (-20°C) ethanol to the tube and place the sample on ice or in a freezer for at least 15 min. You can pause at this stage: under ethanol, purified DNA can theoretically be stored for years. However, based on experience, all three plasmid forms will transition into the linear form.

14. Pellet the plasmid DNA by centrifugation (10 min, 10,000 rpm).

15. Add 1 mL of 70% ethanol to the pellet to wash the DNA precipitate and centrifuge for 3 min.

16. Decant the supernatant, invert the tubes onto a filter, and air-dry the pellet for 0.5 h. Dissolve in 50-100 µL of water. Take 5-10 µL of the sample for Electrophoresis.

17. If necessary, treat the sample with Ribonuclease to remove RNA. Add 1/100 volume of RNase A solution (10 mg/mL in 10 mM Tris-HCl (pH 7.4), 15 mM NaCl) to the resulting plasmid preparation and incubate for 0.5-1 h at 37°C. The RNase can then be removed using phenol, if required. This enzyme does not affect DNA.

Notes

^а Solution II must be freshly prepared. It can be stored only for a limited time, up to several weeks. It is important that during storage, atmospheric CO₂ does not neutralize NaOH. The container must have a tightly fitting screw cap, preferably plastic, since alkalis dissolve Glass. ^б

^б This time is required for lysozyme to act, weakening the peptidoglycan layer. If the enzyme is not added (E. coli cells lyse satisfactorily even without lysozyme), you can proceed directly to the next step.

^в Exceeding the denaturation time runs the risk of irreversibly denaturing the plasmid—one strand will shift relative to the other to such an extent that it reaches a locally stable state. Irreversibly denatured plasmids are quite suitable for sequencing, but they are not cleaved by restriction Enzymes and exhibit anomalous mobility.

^г The alkaline environment is neutralized. Chromosomal DNA rehybridizes randomly, forming large networks, whereas circular plasmid DNA successfully

renatures «back onto itself». It is important that neutralization is complete, leaving no pockets of viscous solution. The solution should be mixed gently at first to avoid fragmenting the genomic DNA, but once association has occurred (after approximately 1 min), vortex more vigorously. Solutions I, II, and III are used in a volumetric ratio of 1:2:1.5 to achieve the desired resulting pH value.