Hello,
I have troubles with genotyping the mutator mouse (PolgAD257A). I use a total DNA extraction kit to extract DNA from mouse tails and then use a hotstart Taq in the genotyping PCR. My other genotyping protocols (not for a mutation in mtDNA, though) work very well. So I wonder if anyone here can share their success in genotyping the mutator mouse. Any thoughts and suggestions would be very much appreciated. Thanks!
Can you give more details about what issues you're having and what conditions you're using? It can be hard to troubleshoot without having more information. The recommended conditions and primers from Jackson labs is found here: https://bit.ly/2NOga1m
That Jackson Protocol that Sofia linked works on the Kujoth et al. (2005) variant, in our hands, as written. It works with dirty extractions, or kit-based DNA extractions. It will also ID the Trifunovic et al. (2004) variant, with a slightly smaller sized band for the "mutator" allele.
Play with your annealing temperatures in case there is some variation in the actual temp in you machine, or try a dilution curve to test different concentrations.