plasmid rescue - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

Plasmid rescue from S. cerevisiae using alkaline lysis or a plasmid mini-prep kit

This protocol allow 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 protocols:

Protocol
alkaline lysis plasmid mini prep
Sigma GenElute™ Plasmid Miniprep Kit,
Thermo Fisher Scientific GeneJET,
Grisp GRS Plasmid Purification Kit
NZYtech NZYMiniprep kit ([[protocol
  1. Spin yeast cells from ~2 ml overnight culture for 10-30 s. Alternatively scrape some biomass from a plate (the fresher the better) and resuspend in 1 mL water.
  2. Spin cells for 20-30 s
  3. Discard supernatant.
  4. Add the amount of resuspension solution to the cells indicated by the kit.
  5. Resuspend cells by vortexing briefly.
  6. Add 200 µL of glass beads, this is about one full 0.2 mL PCR tube. (Optional, add 10 µL lyticase, incubate 20-30 min at 37°C.)
  7. Beat or vortex the tubes for 5 minutes 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.
  8. The disruption of the cells liberate nucleases that can damage the DNA, so add the lysis buffer from the kit as soon as possible, .
  9. If many preparations are made in parallel, put the samples on ice or in an ice-slurry directly after neutralizing the lysis (after third buffer)
  10. Follow the rest of the alkaline lysis miniprep kit protocol.

If the kit has an optional wash step to get rid of nucleases, use that. Elute in a small volume like 30 µL for higher concentration. Transform E. coli with 10 μl of the eluted DNA. Save the rest of the DNA in fridge or at -20°C. This protocol is more laborious than some simpler procedures, but we consistently get more transformants using this protocol.

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.

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