Ron Lab


 

Detecting AIRAP knockout by PCR (XM071 allele)


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