Genotyping Il-10 KO mice has proven difficult, does anyone have any suggestions? Protocols?

Devon Chisholm @Devon-Chisholm

19 September 2016 1 9K Report

Using Common, mutant, and wildtype primers. Getting non-specific bands..but a decent mutant band.

Laurence Stuart Dawkins-Hall

Perform a gradient PCR in which you vary your annealing temp in 1C intervals from Tm-3C to Tm+2C
Alternatively and more directly take your existing annealing temp and increase in 1C intervals...up to Tm + 2C: Hopefully at one of these elevated temp you will retain your specific band but eliminate your non specific band
Reducing your primer concentration from 10uM to 2.5uM might also help
If you add Magnesium to your taq buffer separately reducing your Mg concentration by 0.5mM can also help eliminate non specific bands: Say from 2mM to 1.5mM or 1.5mM to 1mM. Do not go below 1mM otherwise your PCR efficiency is likely to be too low impacting on all bands including specific products

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