Kirsch Lab

Protocols

p53 FRT Genotyping

Schematic of the allele:

 
 
 
 
 
 
 
 
 
Primers:
#1: 5’-CAAGAGAACTGTGCCTAAGAG-3’
#2: 5’-CTTTCTAACAGCAAAGGCAAGC-3’
#3: 5’-ACTCGTGGAACAGAAACAGGCAGA-3’
 
Reaction Components
Reagent Volume (ul)
10x buffer  2
10 mM dNTP mix 0.4
20 uM F Primer  0.4
20 uM R Primer 0.4
Taq DNA Polymerase  1
Nuclease free dH2O 11.8
Genomic DNA (~200 ng)  4
Total 20
 
Cycling:
p53FRT (Primers #1 and #2) 
 
Step Temp (C) Time
1 94 3 min
2 94 30 sec
3 55 30 sec
4 72 40 sec
Back to step 2, 38 times    
5 72 2 min
6 4 hold

Expected Results:
p53FRT
Mutant = 292 bp
Heterozygote = 292 and 258 bp
Wild Type = 258 bp

p53Δ2-6 (Primers #1 and #3)

Step Temp (C) Time
1 94 3 min
2 94 30 sec
3 55 45 sec
4 72 2 min
Back to step 2, 38 times    
5 72 2 min
6 4 hold

Expected Results:
p53Δ2-6:
Recombined allele = 352 bp
Unrecombined or WT = No band

Mouse codon-optimized Flp (FlpO) is described in the following reference:
Raymond, C.S., and Soriano, P. (2007). High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS One 2, e162.