PCR sexing of mouse pups using Sry Y-chromosome primers often results in false-negative females (which turn out to be male later). Sry is a single-copy gene, making PCR detection highly sensitive to DNA template dilution and female DNA contamination.

To solve the Sry primer PCR failure, we optimized our protocol by: 1) Designing multiplex primers that amplify a 200bp segment of Sry alongside a 350bp internal housekeeping control gene (like Myog or Actb) to verify lysis and PCR efficiency. 2) Reducing the tail-snip elution volume to 30 microliters to concentrate the DNA template. 3) Adding 2% DMSO to the PCR master mix to help denature the GC-rich Sry promoter regions. These modifications ensure that a missing band indicates a female pup only if the housekeeping gene is present, eliminating false-negative male identification.