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 miniprep kit for E. coli is needed for this procedure. The Low cost E. coli plasmid alkaline lysis mini-prep can also be used.

We have successfully used:

KIT
Sigma GenElute™ Plasmid Miniprep Kit,
Thermo Fisher Scientific GeneJET,
Grisp GRS Plasmid Purification Kit
NZYtech NZYMiniprep kit	[[protocol

1. Spin yeast cells from 1-1.5 ml overnight culture for 10-30s. Alternatively scrape some biomass from a plate (the fresher the better) and resuspend in 0.5 - 1 mL water. spin cells for 10-30s, Discard supernatant.
2. Add the amount of resuspension solution to the cells indicated by the kit. Resuspend cells by vortexing briefly.
3. Add 200 µl of glass beads (about one full 0.2 mL PCR tube). (Optional, add 10 uL lyticase, incubate 20-30 min at 37C.) Beat or vortex the tubes for 3-5 minutes. 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.

Go to step 4 as soon as possible.

4. Add the lysis buffer as soon as possible, the disruption of the cells liberate nucleases that can digest the DNA. If many preparations are made in parallel, put the samples on ice or in an ice-slurry directly after point three.

5. 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 50 µL or 30 µL for higher concentration.

Transform E. coli with 10 μl of the eluted DNA Save the rest of the DNA at -20C.

This is more laborious than some simpler procedures, but we consistently get more transformants using this protocol.