PCR for mat1

PCR for the active mat1 allele
The mating type is defined by the mating type allele at the active locus mat1. To find the mating type at the mat1 locus we simply do two PCRs, both using:

• primer 46 to the left (centromere proximal) of mat1 as forward (mat1l-K-1-F, 5'-TGGGATGAGTGCTTGCTTTG-3') and with as a reverse
  • primer 66 (Mat-minus-R, 5'-CCACATCTCTCCAACCAGCT-3') yielding a 980bp fragement
  • primer 68 (Mat-plus-R, 5'-CGGTAGTCATCGGTCTTCCA-3') yielding a 1120bp fragment

Do a colony PCR with 60°C as annealing temperature, or use a standard Taq PCR on clean DNA (annealing 58°C).

Alternatively, mating types can be defined by setting up test crosses.