protocol detects the presence of a floxed ATG5 gene (floxed around the 3rd exon).
The mouse strain in our colony was generated by Noboru Mizushima (Tokyo Medical and
Dental University). We are in the process of backcrossing these mice back to BL6 and are
approximately 1 generation away from fully backcrossed mice (6/1/08).
Who designed the protocol:
This protocol was designed by the Mizushima
lab, with the PCR conditions modified
by members of the Virgin lab. The Mizushima lab has also designed a protocol to detect the
rearranged flox allele which I have not included here.
you want to identify the recombinant (Atg5-deleted) band, you need to add the
5L2 primer in
The primer sequence is: 5L2 5'-CAGGGAATGGTGTCTCCCAC-3'.
The recombinant band is 300bp, you can run the samples in 2.5% agarose.
2 ul MgCl2 (25mM)
2 ul dNTPs (2mM)
2 ul 10x TAQ buffer
0.2 ul TAQ polymerase (Promega M166X 19358943)
1 ul Check 2 primer (10uM stock)
1 ul Exon3-1 primer (10uM stock)
1 ul Short2 primer (10uM stock)
1 ul DNA (add 100-300ng)
10 ul dH2O
94°C 4 minutes
94°C 30 seconds
60°C 30 seconds
72°C 1 minute
72°C 5 minutes
Flox allele: 650 bp
Wild Type allele: 350 bp