McClean: Plasmid Loss Assay

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Contents

Overview

This method is appropriate for purposefully losing an undesired plasmid from your strain of interest or for measuring the approximate rate of plasmid loss in non-selective media. Adapted from Lundblad and Zhou, 2001.

Materials

  • Non-selective liquid media (I typically use YPD if I'm dropping a single plasmid, but if you want to drop one plasmid while maintaining others, make sure the media allows for selection of the ones you want to maintain)
  • Non-selective solid media (Same consideration as above.)
  • Selective solid media (Media that should select for the plasmid you're trying to lose.)


Protocol

  1. Grow an overnight culture of a single colony of the plasmid containing strain in non-selective liquid media.
  2. The next day, grow a subculture of this overnight in the same non-selective media to mid-log phase.
  3. Measure OD600 of this culture. Aliquot a small portion and dilute it to OD600 = 0.2.
  4. Take this culture and perform dilutions so that you have 1:1000, 1:10,000, and 1:100,000 dilutions.
    • You'll need 200μL of these dilutions for each plate you plan on plating. You're aiming for ~100 colonies per plate. I like to plate three plates for each dilution.
    • If you don't want to do so much plating, just try the 1:10,000 dilution and adjust if necessary.
  5. Plate 200μL of the dilutions on non-selective plates. Grow at 30°C for 1-2 days. (I like to wait 2 days so I can have a clear indication of the colony concentration. You may have to add another day if using synthetic media.)
  6. Replica these plates onto the selective media plates. Grow all of these at 30°C for 1-2 days, again depending on media composition.
  7. Take each plate and compare. Look for colonies that grew on non-selective media but did not on selective. You can take pictures and use software, such as ImageJ, to compare plates and count colonies if necessary.

Notes

Please feel free to post comments, questions, improvements, or especially clarifications to this protocol.

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References

Lundblad, V. and Zhou, H. 2001. Manipulation of Plasmids from Yeast Cells. Current Protocols in Molecular Biology. 39:13.9.1–13.9.6.

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