Detection of AIRAP knockout allele and wild-type allele by three-primer PCR analysis

Description of mutant allele:

This allele of AIRAP was created by the Baygenomics consortium as a targeting event in the ES line XM071. It is described in greater detail in a publication: Stanhill A, Haynes CM, Zhang Y, Min G, Steele MC, Kalinina J, Martinez E, Pickart CM, Kong X.-P, and Ron D. 2006. An arsenite-inducible regulatory particle-associated protein (AIRAP) adapts proteasomes to proteotoxicity. Molecular Cell 23:875-885.

Note:

Primers:
LacZ.6AS (17/33 GC content): 5’ GGA TGT GCT GCA AGG CGA TTA AGT TGG GTA ACG 3’
mAIRAP.4AS (15/27 GC content): 5’ ACC CAT GTA AGA GCC AGG CAC TTG CAC 3’
mAIRAP.5s (14/27 GC content): 5’ GGG CGG AAT GGA TTT GCT GAT CAA GAG 3’

Products:
~578 bp fragment for WT with mAIRAP.4AS vs. mAIRAP.5s
~311 bp fragment for Mutant with mAIRAP.5s vs. LacZ.6AS

PCR Conditions:
94¾ C, 4 min.
(94¾ C, 30 sec.; 68¾ C, 30 sec.; 72¾ C, 1 min.) x 35 cycles

Buffer:
1.5mM MgCl2 (Fisher) or homemade 10x buffer (100mM Tris pH 8.3, 500mM KCl, 15mM MgCl2