Animal husbandry and handling

For genotyping R26-CAG-LSL-Sun1-sfGFP-Myc+/+ we used a modified version of the JAX protocol. However, this genotyping only tells you that the transgene is there, and if it is homozygous or heterozygous.  I have not seen any protocol for genotyping to detect if the LSL is recombined, though it should be possible to design if one had the transgene sequence. This may be available through Jeremy Nathans' group at Johns Hopkins, who developed the mice.

For our genotyping, we monitored recombination using the other allele (Dnmt3a flox/flox). In this genotyping scheme we can detect the recombined allele.

Here is the genotyping scheme used for the R26-CAG-LSL-Sun1-sfGFP-Myc+/+ allele (again without the ability to see LSL status). Still this may be useful as it is a single reaction as opposed to 2 separate proposed by JAX.

Primers:

Mix primers with 20uM 16014 + 10uM each 16015 and 8820

For a 1X reaction:

6uL EcoTaq (or equivalent)

5uL H2O

1uM primer mix

1.5uL Tail lysis/DNA

Cycling

95C 5min

94C 30sec

56C 40sec

72C 1min

go to step 2 34 times

72C 5min

4C forevver

Band size:

WT = 500bp

Transgene = 300bp

Category