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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 decoment 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).
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