Plasmid rescue from S. cerevisiae using alkaline lysis or a plasmid mini-prep kit
This protocol allows the isolation of small quantities of plasmid DNA from Saccharomyces cerevisiae for the subsequent transformation of E. coli (i.e. plasmid rescue). An alkaline lysis protocol or miniprep kit for E. coli is needed for this procedure. We have successfully used the following protocol and kits:
| Protocol |
|---|
| alkaline lysis plasmid mini prep |
| Sigma GenElute™ Plasmid Miniprep Kit |
| Thermo Fisher Scientific GeneJET |
| Grisp GRS Plasmid Purification Kit |
| NZYtech NZYMiniprep kit (protocol) |
- Spin down yeast cells from ~2 mL of overnight culture for 10-30 s. Alternatively, scrape some biomass from a plate (the fresher, the better) and resuspend it in 1 mL of water.
- Spin down the cells for 20-30 s.
- Discard the supernatant.
- Add the amount of resuspension solution specified in the kit instructions to the cells.
- Resuspend the cells by vortexing briefly.
- Add 200 µL of glass beads, equivalent to about one full 0.2 mL PCR tube. (Optional: add 10 µL of lyticase and incubate for 20-30 min at 37°C.)
- Beat or vortex the tubes for 5 min in a disruptor genie. For large batches of tubes, use LBM big vortex for 5 min at 2500 rpm. Using a FastPrep device might improve yields compared to a vortex.
- Disrupting the cells releases nucleases that can damage the DNA, so add the lysis buffer from the kit as soon as possible.
- If many preparations are made in parallel, put the samples on ice or in an ice slurry immediately after neutralizing the lysate (after adding the third buffer).
- Follow the rest of the alkaline lysis miniprep kit protocol.
If the kit has an optional wash step to remove nucleases, use it. Elute in a small volume, such as 30 µL, to obtain a higher DNA concentration. Transform E. coli with 10 µL of the eluted DNA. Store the remaining DNA in the fridge or at -20°C. This protocol is more laborious than some simpler procedures, but we consistently obtain more transformants with it.
Singh, Madhu V, and P Anthony Weil. 2002. “A Method for Plasmid Purification Directly from Yeast.” Anal. Biochem. 307 (1): 13–17. https://doi.org/10.1016/S0003-2697(02)00018-0.