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Issues with multiplex PCR genotyping

PCR bands genotyping

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#1 whitann06



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Posted 11 October 2013 - 05:32 AM

I am having a lot of issues with genotyping a particular mouse line in our lab.  We obtained the mice from Jackson labs, and have always used their genotyping assay and primers, which have been fine for the past 4 years.  Recently I have been trying to collect mutant embryos by breeding 2 heterozygous mice together, and was not able to get any mutants according to PCR genotyping.  So, I went back and had a company regenotype all of my breeders to make sue they were hets.  Every single one came back as a WT, meaning that I have lost the line, and it is no longer available for purchase.  We are stopping the experiments, but I need to know where things went wrong when I defend my thesis next month.  

We think that the problem was in the PCR design.  It is a multiplex PCR, with 3 primers, that gives a WT and a mutant band which are only about 100 bp apart.  Hets will have both bands.  So when I see mice that genotype having both bands, I kept them thinking they were hets, which they apparently were not.  When I ran the original genotyping for these, there are 2 bands.  But when the company does it there is only a WT.  Are there other things that the second band could have been when I ran the genotyping (shadow or stutter)?  

Thanks for any help! 

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