Ron Lab


 

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

232 bp fragment for WT with PERK.i6AS vs. mPERK1730S

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


PCR Conditions

94˚C, 4 min. (94˚C, 30 sec.; 64˚C, 30 sec.; 72˚C, 30 sec.) x 35 cycles

72˚C, 10 min.

4˚C, 30 min.


RT

Sequence of the 232 bp fragment for WT with PERK.i6AS vs. mPERK1730S PCR fragment:

AAGGACCCTA TCCTCCTGCT GCACTGGTGG AAGGAGATAT TCGGGACGAT CCTGCTTTGC ATCGTAGCCA CGACCTTCAT CGTGCGCAGG CTTTTCCATC CTCAGCCCCA CAGGGTAAGA TGCTCTGTCA ACCTAATGTG CTTCCAAGTG GTTGCTGTGT AGGAAACCTC TGGGAGGGAG AGTTCCGGCA CTCAGACTTA CCAGAGAATC ATCTGCGCTT GTACTGTCTC CG

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

CGGAGACAGT ACAAGCGCAG ATGATTCTCT GGTAAGTCTG AGTGCCGGAA CTCTCCCTCC CAGAGGTTTC CTACACAGCA ACCACTTGGA AGCACATTAG GTTGMCAGAG CATCTTACCC TGTGGGGCTG AGGATGGAAA AGCCTGCGCT CGAGAATTCG TCGAGGGCCC CTGCAGGTCC TACCGGGTAG GGGAGGCGCT TTTCCCAAGG CAGTCTGGAG CATGCGCTTT SGCAGCCCCG CTGGCACTTG GCGCTACACA AGTGGCCTCT GGCCTCGCAC ACATTCCACA TCCACCGGTA GC