Use of M13KO7 Helper Phage for isolation of single-stranded phagemid DNA
- Transform phagemid vector into appropriate F' strain (CJ236 for Kunkel mutagenesis).
- Inoculate 50 ml LB (phagemid antibiotic only) with a fresh colony, grow at 37°C, 250 rpm* until just slightly turbid (<10 Klett, A600 < 0.05). For Kunkel mutagenesis, add uridine to 0.25 µg/ml. *Vigorous aeration is required, i.e. shaking at 250rpm and using a flask with the capacity of at least 5x the culture volume.
- Add 50 µl M13KO7 helper phage (final concentration of 1 x 108 pfu/ml) and continue shaking 60-90 minutes.
- Add kanamycin to final concentration of 70 µg/ml, grow overnight (14-18 hours) at 37°C, 250 rpm.
- Spin culture at 4,000xg for 10 minutes. Transfer supernatant to a new tube and repeat spin.
- Pipet the upper 90% of supernatant into a new tube. To this supernatant, add a 0.2 volume of 2.5 M NaCl/20% PEG-8000. Gently mix several times. Incubate at 4°C for 60 minutes.
- Recover the phage by centrifugation at 12,000xg for 10 minutes. Carefully, decant supernatant and spin again briefly.
- Gently, resuspend the pellet in 1.6 ml TBS. Aliquot into 2 microfuge tubes.
- Spin in a microfuge for 1 minute to pellet any remaining cells, transfer supernatant to new tubes.
- Add 160 µl of the 2.5 M NaCl/20% PEG-8000 solution to each, let sit at room temperature for 5 minutes, spin in a microfuge for 10 minutes at high speed.
- Decant the supernatant, spin again briefly, remove last traces of supernatant with pipetman.
- Resuspend each phage pellet in 300 µl TE. Extract with phenol (let sit 15 minutes before spinning), then phenol/chloroform (50/50: v/v; twice), and finally chloroform. Add 30 µl 2.5 M sodium acetate, pH 4.8 and 2-2.5 volumes ethanol to precipitate at -20°C for ~2 hours.
- Suspend the dried pellets in 25-50 µl TE. Check yield by DNA gel. Helper phage genome (~8660bp) will appear in small amounts relative to phagemid DNA. Single-stranded DNA stains less brightly with ethidium bromide and migrates differently than double-stranded DNA. Yield should be >50 µg single-stranded phagemid for pUC origin vectors.
NEB does not recommend the use of M13KO7 as a cloning vector. For display of short peptides on pIII coat protein of M13, we recommend the Ph.D. Cloning System (#E8101S) with the M13KE phage vector.