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Methods:CTAB M13 phagemid prep

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Contents

Purpose

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Preparation of ssDNA from M13/[Phage_f1|[f1]]-based phagemids

Materials Needed

  • M13 rv-1 helper phage
  • phagemid-containing cells (e.g. from pZ150 or pZ152[1])
  • 2X YT media
  • PEG Solution
    • 20% PEG 8000
    • 2.5 M NaCl
  • CTAB solution
  • 0.3 M Na Acetate pH 5

Protocol

Phage prep

  1. Grow phagemid containing cells overnight in 2XYT
  2. spin 10 minutes in a microfuge in a 1.5 ml tube
  3. transfer supernatant to a fresh microfuge tube

DNA prep

  1. mix 1.2 ml of the phage prep with 0.3 ml PEG solution. Incubate > 30 min (overnight in the cold works)
  2. precipitate the phage
    • Spin 5-10 min in the microfuge.
    • Aspirate off supernatant.
    • Respin briefly to collect remaining liquid; aspirate this off, taking care to not dislodge the pellet, which should be about the size of a pinhead. Pellets can be stored frozen
  3. Resupend in 0.3 ml CTAB buffer. Votex vigorously
  4. Spin 15 minutes, respin and remove residual liquid
  5. Resuspend in 0.1 ml Na Acetate.
  6. Ethanol precipitate

Usage Examples

This prep was obtained from the Youderian lab and used in the Sauer lab in the 1990s for dideoxy sequencing. The phage stocks can also be used for Method:M13-mediated phagemid transduction

User notes

References

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  1. Zagursky, RJ & Berman, ML (1984) Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene 27 183-91 PubMed EcoliWiki page