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

Isolation of Plasmid and Phage DNA
Isolation of M13 Single-Stranded Phage DNA

Phage particles (virions) released from infected E. coli Cells into the culture medium are relatively easy to isolate. First, the cells are removed by centrifugation, and the phage (as well as phagemid) particles are precipitated from the culture medium using polyethylene glycol (PEG).

The single-stranded linear DNA molecule of each virion is enclosed within a protein capsid. The wild-type phage DNA is 6,407 nt in size. The capsid consists of approximately 2,700 molecules of the gp8 coat protein and a few molecules of gp3, gp6, gp7, and gp9 Proteins, which assemble onto the +-DNA strand during phage particle self-assembly. The particles measure 895 nm in length and 6 nm in diameter. The Proteins can be denatured and subsequently removed using phenol or other chaotropic agents that disrupt the Tertiary Structure of macromolecules, after which the released DNA is precipitated from the solution with ethanol. The yield of phage DNA obtained from a 1.5 mL culture medium of infected E. coli is sufficient for Electrophoresis and sequencing.

Materials and Equipment

E. coli culture infected with M13 K07 phage, orbital shaker.

Solutions

- PEG/NaCl solution. Aqueous solution of 20% PEG-6000; 2.5 M NaCl.

- Phenol-chloroform mixture (Procedure 1.1).

- Chloroform-isoamyl alcohol mixture (Procedure 1.1).

Procedure

1. Transfer 10 µL of phage-infected E. coli cells from a culture grown to the logarithmic growth phase in a liquid nutrient medium to 2.5 mL of 2YT nutrient medium containing an antibiotic ^a. Alternatively, a single colony can be picked with an inoculation loop from a fresh 2YT Agar plate.

2. Incubate the culture overnight in a shaking incubator at 200–300 rpm and 37°C with vigorous aeration.

3. Take 1.5 mL of the culture and transfer it to a 1.7 mL Eppendorf tube.

4. Pellet the cells by centrifugation for 5–10 minutes.

5. Transfer the supernatant to a fresh tube and add 1/10 volume of PEG/NaCl (150 µL) to precipitate the phages. Leave at 0°C for 15 minutes.

6. Centrifuge for 5 minutes at maximum microcentrifuge speed.

7. Remove the supernatant using a micropipette.

8. Resuspend the phage particle pellet in 400 µL of TE buffer ^b.

9. Add an equal volume of the phenol-chloroform mixture, mix thoroughly by vortexing, and centrifuge for 5 minutes. This extracts the DNA from the DNP complex. The DNA remains in the upper aqueous phase, phenol forms the lower phase, and denatured proteins accumulate at the interphase.

10. Carefully collect approximately 300–350 µL of the upper aqueous phase into a clean tube, taking care not to disturb the interphase.

11. Add an equal volume of the chloroform-isoamyl alcohol mixture, vortex, and centrifuge for 5 minutes. Phase Separation occurs, and residual phenol dissolves in the lower organic phase.

12. Transfer approximately 250–300 µL of the aqueous phase to a clean tube.

13. Add 1 mL of 96% ethanol and centrifuge for 10 minutes at 13,000 rpm.

14. Discard the supernatant and wash the pellet. Dissolve the DNA in 10 µL of H₂O.

Notes

^a Selection against uninfected Bacteria is strictly required. Unlike lambda phage, M13 family phages do not lyse the cells they infect. They replicate, package into virions, and exit the cells without halting their division; infected bacteria merely grow and divide 30–40% slower. However, the metabolic burden on the bacterial Cell is so substantial that any uninfected cell will rapidly outgrow and outcompete the infected ones in proliferation.

^b At this stage, you can pause by placing the preparation in the refrigerator. The next day, purify the DNA from protein capsid components by denaturing the proteins with phenol, which disrupts the tertiary STRUCTURE OF THE macromolecule (globule-to-coil transition).