genotyping knockout mice - (Feb/03/2007 )
I have been trying to genotype mice deficient for a particular gene. I find that they are either wild type, heterozygous or knock-out. However, with some samples no band appears in the PCR. The DNA is intact, the quality is good, and the housekeeping gene amplifies well.
Is it possible to lose the wildtype gene through the breeding generations. Is it possible to have a mutation generated in the wildtype gene, hence no amplification?
I cannot find an explanation to the lack of amplification.
perhaps you could try PCRing further back from the point of interest.
How about the primers, are they fine. Could you use fresh primers.
Even if there is a mutation, you should still have a PCR product.
Maybe the samples which show no PCR product are knock outs?