Strain Protocols

Strain Information
Strain Code: 01XAD
Strain Name: B6;129-Gadd45atm1Ajf/Nci
Gene Symbol: Gadd45a
Protocol Information
Allele: Gadd45a<tm1Ajf>
Protocol Number: 1
Introductory Comment: This strain contains a PGK-neo cassette, replacing exons 1-3 and upstream regulatory sequence (3.65 kb sequence in total). The genotyping strategy is to detect the PGK element with primers G45a-mut and G45a-com, while G45a-wt and G45a-com detect the wild-type allele, allowing one to determine zygosity.

Primers

G45wt : 5'-cct ctg ctt acc tct gca caa-3'
G45cm : 5'-gaa gac cta gac agc acg gtt-3'
G45mt : 5'-aga acg aga tca gca gcc tct-3'
Diagram showing the primer relataionships for Strain 01XAD.  This strain contains a PGK-neo cassette, replacing exons 1-3 and upstream regulatory sequence (3.65 kb sequence in total).  The genotyping strategy is to detect the PGK element with primers G45a-mut and G45a-com, while G45a-wt and G45a-com detect the wild-type allele, allowing one to determine zygosity.

Product Sizes

Primer Combination Product Size
G45wt / G45cm 324bp    Wild-type
G45mt / G45cm 211bp    Mutant (PGK-neo)

Master Mix

Reagent Final Concentration
10X Buffer 1X
25mM MgCl2 2.0mM
2.5mM dNTP Mix 0.2mM
100 µMG45wt 0.5 µM
100 µMG45cm 0.5 µM
100 µMG45mt 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 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.