Ron Lab


 

Ire1α 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