I have been trying to genotype my 5xFAD mouse ordered from Jackson lab. For PCR, I use tail samples and Primers ordered from Jax website for those mice. I am not getting consistant results. Heterozygous does not seem to be convincing, I see faint bands and sometimes even my negative control (which is WT and should have only one band) gives faint heterozygous (double) band. I have tried changing primer conc., diferent Taqs, increasing Tm, changing to different batch of H2O. If anyone had also similar problem, please suggest me what should I do !
If your positive control for normal is giving you a het signal then you must run a no template control. If this shows a het genotypr then you have pcr contamination of a het amplimer in your reagent mix or in your working area,
A picture would help to see what the problem looks like
I will definitely do this next time i run PCR and let you know with the pic.. Thanks Paul Rutland !
Try to run individual reactions for wild-type and transgenic mice rather than a single reaction.
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