*Keep all reagents on ice including while adding the DNA. Set up reactions as quickly as possible. Put samples into preheated block. This is a very fussy reaction. We have found if too many reactions are set up at one time the first reactions may fail. Set up independent duplicate reactions with second set set up in reverse order from first set.
**Cycling conditions: We use an MJ Tetrad 96 well block. PUT SAMPLES INTO PREHEATED BLOCK.
To visualize on a gel add 4.5 µl of loading buffer (12% ficoll 400, 0.2% bromophenol blue, 0.04 M EDTA) diluted 1:4 with TEN (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 10 mM NaCl) to 3 µl PCR reaction. Load 7 µl on the gel. Separate on 2 % SeaKem agarose gel with 0.5 x SYBR Green I Nucleic Acid Stain.
Samples sized on ABI 3730.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.