Strain Protocols

Strain Information
Strain Code: 01XA6
Strain Name: STOCK Tgfb1tm1Doe Rag2tm1Fwa/Nci
Gene Symbol: Rag2
Protocol Information
Allele: Rag2<tm1Fwa>
Protocol Number: 1
Introductory Comment: In addition to the targeted mutation in the Tgfb1 gene, this strain carries a targeted insertion of the Neo cassette in the Rag2 gene. This genotyping protocol will distinguish between wt, heterozygote, and homozygote animals carrying the Rag2 mutation. This protocol should be used in addition to the allele specific PCR for the Tgfb1 mutant allele to select double mutant animals.

Primers

R011 : 5'-ggg agg aca ctc act tgc cag ta-3'
R012 : 5'-agt cag gag tct cca tct cac tga-3'
R013 : 5'-cgg ccg gag aac ctg cgt gca a-3'
Diagram showing the primer relataionships for Strain 01XA6.  In addition to the targeted mutation in the Tgfb1 gene, this strain carries a targeted insertion of the Neo cassette in the Rag2 gene.  This genotyping protocol will distinguish between wt, heterozygote, and homozygote animals carrying the Rag2 mutation.  This protocol should be used in addition to the allele specific PCR for the Tgfb1 mutant allele to select double mutant animals.

Product Sizes

Primer Combination Product Size
R011 / R012 263 bp    WT
R013 / R012 350 bp    Mutant (neo)

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0 mM
2.5mM dNTP Mix 0.2 mM
100 µMR011 0.5 µM
100 µMR012 0.5 µM
100 µMR013 0.5 µM
5U/┬Ál Taq Pol 0.015 U
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 0:30
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.