[EXTERNAL] [Zbrafish] Nacre primers

[James Lister]

Hi Cameron,

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.

Cheers,
Jim

On Feb 7, 2018, at 1:11 PM, Cameron Wyatt <***@igmm.ed.ac.uk<mailto:***@igmm.ed.ac.uk>> wrote:

Dear all,

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.

Regards,
Cameron
--
Cameron Wyatt
Zebrafish technologist and manager
HGU fish facility
Institute of Genetics and Molecular Medicine
University of Edinburgh
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.
_______________________________________________
Zbrafish mailing list
***@net.bio.net<mailto:***@net.bio.net>
http://www.bio.net/biomail/listinfo/zbrafish

————
James Lister, Ph.D.
Department of Human and Molecular Genetics
Virginia Commonwealth University School of Medicine
Richmond, VA 23298 USA
***@vcuhealth.org<mailto:***@vcuhealth.org>

[James Lister]

Hi again Cameron,

Also, the 19bp deletion in roy is actually in the mpv17 transcript (due to mis-splicing) not in the genomic DNA. To my knowledge the mutation itself has not been identified, so you’ll have to use non-complementation or sequencing from cDNA to ID those carriers.

-Jim

On Feb 7, 2018, at 1:11 PM, Cameron Wyatt <***@igmm.ed.ac.uk<mailto:***@igmm.ed.ac.uk>> wrote:

Dear all,

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.

Regards,
Cameron
--
Cameron Wyatt
Zebrafish technologist and manager
HGU fish facility
Institute of Genetics and Molecular Medicine
University of Edinburgh
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.
_______________________________________________
Zbrafish mailing list
***@net.bio.net<mailto:***@net.bio.net>
http://www.bio.net/biomail/listinfo/zbrafish

————
James Lister, Ph.D.
Department of Human and Molecular Genetics
Virginia Commonwealth University School of Medicine
Richmond, VA 23298 USA
***@vcuhealth.org<mailto:***@vcuhealth.org>