To begin, inoculate two tubes containing three milliliters of YPD media with the synthetic rho minus cells and the acceptor yeast cells. The next day, transfer 750 microliters of the donor strain and 250 microliters of the receptor strain into a sterile microtube. After centrifuging the mixture for one minute at 13, 200G, transfer a drop of the cell pellet onto a YPD plate and incubate.
Check the culture under a light microscope for the presence of shmoos. If shmoos are present, re-suspend a small amount of the cell mass in two milliliters of YPD medium. Incubate the culture for two hours at 30 degrees Celsius in a rotating wheel.
Vortex the mixture well. Then dilute 10 microliters of the cell suspension in 990 microliters of sterile water. Spread 100 microliters of the diluted culture on a drop-out medium that facilitates the growth of only the acceptor strain.
Replicate the resulting colonies on the necessary selective media. After identifying positive colonies, re-streak them on the same selective media to purify the haploid strain carrying the mitochondrial DNA of interest.
