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

Isolation of Plasmid and Phage DNA
Large-Scale Isolation of Plasmid DNA (Maxiprep)

This method is designed to yield significant quantities of high-quality plasmid DNA, which is often required for extended, large-scale work with specific vectors, recombinant constructs, or cloned genes. The Procedure outlined below is also a Modification of the standard alkaline lysis method for bacterial Cells. In a sense, the DNA isolation procedure simply involves proportionally scaling up The amount of starting material and the volumes of the reagent solutions.

The aim of this work is to obtain valuable vector DNA in substantial quantities. The pBluescript II KS+ phagemid is a vector from StratageneTM used for cloning, sequencing, and expression. Furthermore, unlike conventional Plasmids, this phagemid (derived from phage + plasmid) can be "packaged" into phage particles in the presence of a helper phage in The Cell, as it contains the M13 phage ori, while the helper supplies the phage Proteins required for virion assembly. The pBluescript II vector is characterized by a higher copy number per cell than the pBR322 plasmid (see App. 1, 5).

Materials and Equipment

Large-capacity centrifuge, balance, Escherichia coli XL1-Blue strain containing the pBluescript II KS+ phagemid.

Solutions

- Solution I (Experiment 4.1).

- Solution II (Experiment 4.1).

- 10 M ammonium acetate. Dissolve 770 g in 800 ml of Water and bring the volume to 1 l.

- Ampicillin (Experiment 4.1).

Procedure

1. Grow an overnight bacterial culture at 37°C in 100–500 ml of rich 2YT nutrient medium containing 100 µg/ml ampicillin for 20–24 h on a rotary shaker-incubator at 200–300 rpm.

2. Centrifuge at 4000 rpm for 10–15 min at 4°C, then discard the medium.

3. Centrifuge at 4000 rpm for 1 min and carefully remove any residual liquid with a micropipette.

4. Resuspend the pelleted cells in 10 (or 5) ml of Solution I.

5. Add 1 ml (or 0.5 ml) of freshly prepared Solution I containing Lysozyme. Let stand at room Temperature for 15 min. (E. coli cells will also lyse in the absence of lysozyme).

6. Add 20 (or 10) ml of Solution II and mix vigorously right away. Place on ice at 0°C for 5 min.

7. Add 10 (or 5) ml of cold 10 M ammonium acetate ^a dropwise using a micropipette, and mix. Incubate at 0°C for 5 min.

8. Centrifuge at 5000 rpm for 10 min at +4°C.

9. Collect the supernatant with a micropipette, divide it equally between two centrifuge tubes, and add 12.5 ml of isopropanol to each. Balance the tubes on a scale. Let stand at room temperature for 10 min to precipitate the DNA.

10. Centrifuge at 5000 rpm at 4°C for 10 min, then discard the supernatant.

11. Centrifuge at room temperature for 3 min and remove any residual liquid. Do not let the plasmid DNA pellet dry out!

12. Dissolve the pellet in each microcentrifuge tube in 400 µl of 2 M ammonium acetate, and combine them into a single 1.7 ml tube. Let stand at room temperature for 5 min (to precipitate Polysaccharides and proteins).

13. Centrifuge at room temperature for 10 min.

14. Transfer the supernatant to a tube containing an equal volume (800 µl) of isopropanol ^b. Leave on the bench for 10 min.

15. Centrifuge at room temperature for 5 min.

16. Wash the pellet with 70% ethanol.

17. Dissolve the plasmid DNA in 0.2–1 mL of H₂O. For Electrophoresis, 5–10 µL of the solution ^c is sufficient.

Notes

^a Using ammonium acetate instead of potassium acetate for solution III significantly reduces centrifugation volumes. In addition, ammonium acetate precipitates polysaccharides and proteins from the solution (see step 12). This makes it possible to omit the phenol extraction step from the protocol.

^b Using isopropanol to precipitate DNA from solution helps reduce volumes. In large-scale Procedures, It is important to minimize reagent consumption. Typically, an equal volume of isopropanol is used (at least 0.6 V, compared to 2 V for ethanol).

^c The electrophoretic mobility of Various Forms of small plasmids is given in Appendix 9. The supercoiled form migrates first; at very high concentrations, a supersupercoiled form can be seen ahead of it. This is followed by the linear form (found in older preparations or when protocols are violated) and then the relaxed plasmid form (a covalently closed circular duplex with a nick in one strand). These are the three main forms. Above these forms in concentrated preparations, a dimeric supercoiled form may be observed. Even higher, toward the well, chromosomal DNA remnants are sometimes visible. Upon restriction Digestion, all forms are converted into a single band corresponding to the linear form (remnants of the host strain chromosome produce a smear of fragments that is usually invisible).