Actually w2 is a substitution that fortuitously creates a Dra I restriction site, so the genotyping is pretty straightforward and you can use pretty much any primers to your liking that flank the mutation (although be aware there is another Dra I site not too far away.) I can send you our primer info/protocol if you like.
On Feb 7, 2018, at 1:11 PM, Cameron Wyatt <Cameron.Wyatt from igmm.ed.ac.uk<mailto:Cameron.Wyatt from igmm.ed.ac.uk>> wrote:
Does anyone have experience genotyping nacre mutants (mitfa^w2) by PCR?
I have pigmented offspring of casper hets that I would like to genotype. Designing primers for the roy mutation (mpv17^a9) appears to be easy enough as it is a 19bp deletion. However the nacre mutation is a 1bp substituition. I've never used PCR to identify a 1bp mutation and I've read that 'sometimes it works, sometimes it does not'.
Does anyone know if PCR works for this particular mutation (presumably with a primer 3' aligned with mutant bp)?
To save people the time of writing to suggest alternatives, I will say that I know can sequence the region containing the mutation or pair mate the fish with homo caspers instead.
Thanks for any advice.
Zebrafish technologist and manager
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Institute of Genetics and Molecular Medicine
University of Edinburgh
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James Lister, Ph.D.
Department of Human and Molecular Genetics
Virginia Commonwealth University School of Medicine
Richmond, VA 23298 USA
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