Ron Lab


 

Detection of ATF4 knockout allele and wild-type allele by four-primer PCR analysis

Description of mutant allele

Detection of mouse ATF4 mutant allele (Jeff Leiden's) and wild-type alleles by 4-primer PCR analysis. This ATF4 allele has been described in Hettman et al 2000 Dev Biol 222:110-123

Note:
These mice were used to derive MEFs in: Harding H, Zhang Y, Zeng H, Novoa I, Lu P, Calfon M, Sadri N, Yun C, Popko B, Paules R, Stojdl D, Bell J, Hettmann T, Leiden J and Ron D. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Molecular Cell 11:619-633 (2003). A pdf version of this document with a cartoon of the mutant and wildtype alleles is available for downloading.


Primers

mATF4.5S

TTT GAG TTG TGC GCT CGG GTG TC

PGK.255R

GCT ACC GGT GGA TGT GGA ATG TG

mATF4.7S

GCA GCA GTG TTG CTG TAA CGG AC

mATF4.6AS

CT GGC CAA GCC ATC ATC CAT AGC


Products

ATF4Leiden mutant allele (mATF4.5S vs. PGK.255R) ~400bp Wild type allele (mATF4.7S vs. mATF4.6AS) 277bp


PCR Conditions

  • Denature 94 4min 1 cycle
  • Denature 94C 30 sec
  • Anneal 64C 30 sec 30 cycles
  • Extend 72C 1min
  • Extend 72C 10min 1 cycle

PCR reaction instructions

In a total volume of 25 ml per reaction: Use 1ml each of pGK255R, mATF4.5S, mATF4.7S, mATF4.6AS (10pmol/ml stock) per reaction. Use 2.5 ml of 10x PCR assay buffer (500mM Tris pH9.0, 200mM NH4SO4, 15mM MgCl2). Use 1.25 ml of 10x stock of dNTPs (2.5mM).