Strain Protocols

Strain Information
Strain Code: 01XMD
Strain Name: B6;129S-Mdm4GT(VICTR20)7Lex Mdm2tm1BayTrp53tm1Brd/Nci
Gene Symbol: Mdm2
Protocol Information
Allele: Mdm2<tm1Bay>
Protocol Number: 1
Introductory Comment: Mdm2 allele genotyping protocol: The genoytypes are performed with two separate PCR reactions to obtain mutant allele and wildtype allele information due to tthe similar size of amplifcon and are difficult to resolve in gel. The mutant allele PCR uses primers Y681/Y682 and wildtype allele PCR uses primers Y683/Y684.

Primers

Y681 : 5'-ggc aaa gga tgt gat acg tgg aag-3'
Y682 : 5'-cca gtt tca cta atg aca caa aca tg-3'
Y683 : 5'-gga gat cct gag att tcc tta gc-3'
Y684 : 5'-ggc tct tta gca tca ttc tct gtc-3'

Product Sizes

Primer Combination Product Size
Y681 / Y682 830 bp    Mutant
Y683 / Y683 647 bp    Wild Type

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2
2.5mM dNTP Mix 0.2mM
100 µMY681 0.5 µM
100 µMY682 0.5 µM
100 µMY683 0.5 µM
100 µMY684 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.