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PERK.KO4 genotype by PCR (9/99)

This mutant allele deletes the region encompassing the trans-membrane domain of PERK. It is described in (Harding, et al., 2000, Molecular Cell; 5:897, Harding, et al., 2001, Molecular Cell; 7, 1153-1163)

(see comment on detecting the PERK.KO4 allele in mutant fibroblasts)

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’
PERK.i6AS 13/24 GC content
5’ CGG AGA CAG TAC AAG CGC AGA TGA 3’
mPERK1730S 14/24 GC content
5’ AAG GAC CCT ATC CTC CTG CTG CAC 3’TG

PCR Products:

231 bp fragment for WT with PERK.i6AS vs. mPERK1730S
302 bp fragment for KO with PERK.i6AS vs. PGK.255R

PCR Conditions:
94¾ C, 4 min.
(94¾ C, 30 sec.; 64¾ C, 30 sec.; 72¾ C, 30 sec.) x 35 cycles
72¾ C, 10 min.
4¾, 30’

RT

Sequence of the 231 bp fragment for WT with PERK.i6AS vs. mPERK1730S PCR fragment:

AAGGACCCTATCCTCCTGCTGCACTGGTGGAAGGAGATATTCGGGACGATCCTGCTTTGCATCGTAGCCACGACCTTCATCGTGCGCAGGCTTTTCCATCCTCAGCCCCACAGGGTAAGATGCTCTGTCAACCTAATGTGCTTCCAAGTGGTTGCTGTGTAGGAAACCTCTGGGAGGGAGAGTTCCGGCACTCAGACTTACCAGAGAATCATCTGCGCTTGTACTGTCTCCG

Sequence of the 302 bp fragment for KO with PERK.i6AS vs. PGK.255R PCR fragment:

CGGAGACAGTACAAGCGCAGATGATTCTCTGGTAAGTCTGAGTGCCGGAACTCTCCCTCCCAGAGGTTTCCTACACAGCAACCACTTGGAAGCACATTAGGTTGMCAGAGCATCTTACCCTGTGGGGCTGAGGATGGAAAAGCCTGCGCTCGAGAATTCGTCGAGGGCCCCTGCAGGTCCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTSGCAGCCCCGCTGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGC