Plasmid Recovery from yeast cells

Grow in 5 mL of selective media cells carrying the plasmid overnight at 30 C with shaking.

Centrifuge the overnight culture and discard the supernatant. 

Resuspend the cells in 0.5 mL sorbitol solution (0.9 M sorbitol, 0.1 M Tris-HCl pH=8.0, 0.1 M EDTA) and transfer in a 1.5 mL microcentrifuge tube.

Dip the tip of a sterile toothpick in the zymolyase powder and rotate the toothpick a couple of times. Remove the excess powder from the toothpick by tapping it against the vial as you pull it out.

Dip the tip of the toothpick, now lightly coated with zymolyase powder, into the cell suspension and rotate the toothpick a couple of times to dissolve the powder in the solution.

Incubate with shaking at 37 C for one hour or until spheroplasting is clear and visible (a viscous, stringy appearance of the cell suspension).

Centrifuge briefly to collect the spheroplasts at the bottom of the tube. 

Remove as much of the supernatant as possible without getting too close to the spheroplasts.

Resuspend the spheroplasts in 0.25 mL of buffer 1 of the Qiagen plasmid miniprep kit for E coli. Follow all the steps of the kit's protocol, including the PB resuspension step. The spheroplasts should be fully lysed at the lysis step (buffer 2). Be patient, wait, and incubate the tube at 50 C if necessary.

Once you finish, use 2-5 uL of the yeast plasmid miniprep to transform E coli with the recovered plasmid.