Mating Test Protocol
We used BY4710 (Mat a trp-) and BY4 711 (Mat a
trp-) as mating testers. Mating tests can also be performed with strains
streaked out on plates. After checking strains by patching them out
on their respective drop out plates, the strains can be crossed to
a different auxotroph background strain to test for correct mating
types on SD plates. Diploids should not mate with either A or alpha
tester, therefore serve as negative control.
Transfer strains to be tested from 96-well microtiter plate (MTP)
into a fresh MTP containing 50 to 75 ul YPD in each well using a
96-pin tool .
Streak out the a or a mating testers onto
YPD plate so it makes a coarse lawn.
Incubate MTP and tester plates overnight at 30 °C.
Using sterile velvets and replicating block, transfer tester strain
from YPD plate unto a maximum of 6 SD plates. Change velvet and repeat
for the other tester strain using fresh SD plates.
Check the growth of the deletion strains in the MTP.
Transfer a small amount (less than 1 ul) of liquid culture from MTP
onto A tester plate or alpha tester plate (sterilize the pin tips
by rinsing in water first, followed by ethanol and flaming). Be sure
to include proper controls on the SD plates.
Incubate at 30 °C.
Day 3 to 5:
Check for growth. Colonies are formed only after successful mating
between opposite mating types and complementation of auxotrophy on