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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)
1 94
2 94
3 55
4 72
Back to step 2, 38 times  
5 72
6 4

Expected Results:

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

p53Δ2-6 (Primers #1 and #3)

Step Temp (C)
1 94
2 94
3 55
4 72
Back to step 2, 38 times  
5 72
6 4

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.