To begin, add 1.5 milliliters of 70%ethanol to 30 milligrams of 0.7-micrometer tungsten particles in a microtube. Centrifuge the mixture at 13, 200G for 15 minutes. Add 1.5 milliliters of sterile water to the particles, then vortex and centrifuge again.
After discarding the supernatant, add 500 microliters of sterile 50%glycerol to the tungsten particles. To coat the particles, first add five micrograms of a two-micron plasmid and 15 micrograms of a bacterial plasmid containing the mitochondrial sequence into a microtube placed on ice. Add 100 microliters of the tungsten particle suspension to the tube and vortex.
Then add four microliters of one-molar spermidine and vortex again. Now pipette 100 microliters of ice cold 2.5-molar calcium chloride into the tube. After briefly vortexing the suspension, incubate it on ice for 10 minutes.
Centrifuge the tungsten particles at 13, 200G for 30 seconds at four degrees Celsius. Then, wash the particles in 200 microliters of 100%ethanol at minus 20 degrees Celsius. Finally, re-suspend the tungsten particles in 60 to 70 microliters of room temperature 100%ethanol.
To prepare the biolistic materials, place six sterilized macrocarrier holders on a sterile glass plate. Use a pair of sterile forceps to insert the macrocarriers into each of the six macrocarrier holders. Pipette 10 microliters of the DNA-coded tungsten particles onto each macrocarrier surface and evenly distribute over the entire surface with the pipette tip.
Inoculate the rho zero yeast receptor strain in two milliliters of YPR medium. Grow the culture overnight at 30 degrees Celsius in a rotating wheel. The next day, transfer 300 microliters of the culture into a tube containing 30 milliliters of fresh YPR medium.
Place the culture on a rotary shaker for two nights at 30 degrees Celsius and 200 revolutions per minute until the culture is saturated. Centrifuge the yeast cells at 600G for five minutes at room temperature. Re-suspend the cells in 600 microliters of YPD medium after discarding the supernatant.
Now use a sterile glass handle to spread 100 microliters of the yeast cell suspension on a solid bombardment medium plate. After spreading all the cultures, incubate the plates at room temperature for one to three hours before bombardment.
