Strain Protocols

Strain Information
Strain Code: 01XMB
Strain Name: B6;129S4-Mdm2tm4.1Snj/Nci
Gene Symbol: Mdm2
Protocol Information
Allele: Mdm2<tm4.1Snj>/Nci
Protocol Number: 1
Introductory Comment: The genotyping protocol utilizes two alleleic specific amplifications: one is for mutant allele and the other is for wild type allele. The genotyping results from both PCR is combined to determine the genotypes. Mutant specific PCR: primers Y701 and Y702. Wild type specific PCR: primers Y701 and Y703. The primer concentration used in PCR is 0.5 uM each.

Primers

701 : 5'-GAC GAC TTT TCC CAA CCA TCC-3'
702 : 5'-CCA GTC TTG CCG TGA ACA AT-3'
Y703 : 5'-GAC GAC TAT TCC CAA CCA TCG-3'

Product Sizes

Primer Combination Product Size
701 / 702 202 bp    Mutant allele
Y703 / 702 202 bp    Wildtype allele

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2 mM
2.5mM dNTP Mix 0.2mM
100 µM701 0.5 µM
100 µM702 0.5 µM
100 µMY703 0.5 µM
5U/┬Ál Taq Pol 0.025 U/µl
Use 50 - 100 ng of DNA per reaction

Cycling Conditions

(Time in minutes:seconds)
Temperature Time Label
94ºC 3:00
94ºC 0:30
60ºC 0:30 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.