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  • Use of M13KO7 Helper Phage for isolation of single-stranded phagemid DNA

    Protocol

    1. Transform phagemid vector into appropriate F' strain (CJ236 for Kunkel mutagenesis). 
    2. 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. 
    3. Add 50 µl M13KO7 helper phage (final concentration of 1 x 108 pfu/ml) and continue shaking 60-90 minutes. 
    4. Add kanamycin to final concentration of 70 µg/ml, grow overnight (14-18 hours) at 37°C, 250 rpm. 
    5. Spin culture at 4,000xg for 10 minutes. Transfer supernatant to a new tube and repeat spin. 
    6. 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. 
    7. Recover the phage by centrifugation at 12,000xg for 10 minutes. Carefully, decant supernatant and spin again briefly. 
    8. Gently, resuspend the pellet in 1.6 ml TBS. Aliquot into 2 microfuge tubes. 
    9. Spin in a microfuge for 1 minute to pellet any remaining cells, transfer supernatant to new tubes. 
    10. 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. 
    11. Decant the supernatant, spin again briefly, remove last traces of supernatant with pipetman. 
    12. 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. 
    13. 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.