Ire1a KO genotype by PCR (3/99)

Buffer: 1.5mM MgCl₂ (Fisher) or homemade 10x buffer (100mM Tris pH 8.3, 500mM

KCl, 15mM MgCl₂

Primers:

PGK.255R 13/23 GC content

5’ GCT ACC GGT GGA TGT GGA ATG TG 3’

mIre1a.g4AS 13/22 GC content

5’ GCA AGA GTC AGG CCC ACT GAT C 3’

mIre1a.g5S 13/23 GC content

5’ TTA AGA CCC AGC ACT GGC CAG GT 3’

PCR Products: ~315 bp fragment for KO with PGK.255R vs. mIre1a.g4AS

~400 bp fragment for wt with mIre1a.g5S vs. mIre1a.g4AS

PCR Conditions:

94˚ C, 4 min.

(94˚ C, 30 sec.; 57˚ C, 30 sec.; 72˚ C, 30 sec.) x 30 cycles

72˚ C, 10 min.

4˚, indefinite