Strain Protocols

Strain Information
Strain Code: 01XL9
Strain Name: 129S4-Trp53tm2.1Tyj/Nci
Gene Symbol: Trp53
Protocol Information
Allele: Trp53<tm2.1Tyj>
Protocol Number: 1
Introductory Comment: These mice express a point mutation in the p53 gene under control of the endogenous Trp53 promoter. Line 129S4-Trp53 - AKA P53 R172H (01XL9) - contains a point mutation in Exon 5, converting an Arg into a His. Line 129S4-Trp53 - AKA p53 R270H (01XM1)- contains a point mutation in Exon 8, converting an Arg into a His. These 2 lines contain a leftover LoxP site from recombination of 01XM2 / 01XM3. Therefore, the genotyping strategy is to span the LoxP site to distinguish them from WT. These primers do not distinguish p53 R172H (01XL9) from p53 R270H (01XM1). Sequencing may be needed to confirm the mutation.

Primers

T034 : 5'-ctt gga gac ata gcc aca ctg-3'
T035 : 5'-agc ctg cct agc ttc ctc agg-3'
Diagram showing the primer relataionships for Strain 01XL9.  These mice express a point mutation in the p53 gene under control of the endogenous Trp53 promoter. Line 129S4-Trp53 - AKA P53 R172H (01XL9) - contains a point mutation in Exon 5, converting an Arg into a His. Line 129S4-Trp53 - AKA p53 R270H (01XM1)- contains a point mutation in Exon 8, converting an Arg into a His. These 2 lines contain a leftover LoxP site from recombination of 01XM2 / 01XM3. Therefore, the genotyping strategy is to span the LoxP site to distinguish them from WT. These primers  do not distinguish p53 R172H (01XL9) from p53 R270H (01XM1).  Sequencing may be needed to confirm the mutation.

Product Sizes

Primer Combination Product Size
T034 / T035 290    WT
T034 / T035 330    LoxP

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0 mM
2.5mM dNTP Mix 0.2 mM
100 µMT034 0.5 µM
100 µMT035 0.5 µM
5U/┬Ál Taq Pol 0.015 U/µl
Use 50 - 100 ng of DNA per reaction

Cycling Conditions

(Time in minutes:seconds)
Temperature Time Label
94ºC 3:00
94ºC 1:00
60ºC 2:00 35 Cycles
72ºC 1:00
72ºC 3:00
4ºC HOLD

Disclaimer: PCR conditions were optimized using QIAGEN-purified mouse tail DNA and a PTC-200 thermocycler (MJ Research). Optimal conditions may vary with DNA quality or equipment used.