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
- Grow an overnight culture of a single colony of the plasmid containing strain in non-selective liquid media.
- The next day, grow a subculture of this overnight in the same non-selective media to mid-log phase.
- Measure OD600 of this culture. Aliquot a small portion and dilute it to OD600 = 0.2.
- 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.
- 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.)
- Replica these plates onto the selective media plates. Grow all of these at 30°C for 1-2 days, again depending on media composition.
- 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