Hello everyone,

I am facing a persistent issue with PCR genotyping of mouse tail DNA for the presence of SFTPC-Cre and G12C genes. Our genotyping panel includes three genes, including p53, SFTPC-Cre, and G12C. While PCR reactions for p53 consistently yield clear bands using the same reagents and protocol, SFTPC-Cre and G12C reactions are showing persistent streaking in the gels, including in the no-template controls (NTCs).

Here’s what we have tried so far:

• Ordered and tested new, high-quality primers specifically for SFTPC-Cre and G12C.
• Used fresh aliquots of master mix, extraction buffers, and stabilization reagents.
• Collected new mouse tail samples and performed fresh DNA extractions.
• Reduced the DNA quantity and conducted gradient PCR to optimize annealing temperatures.
• Ensured a clean setup environment by using new reagents, filtered tips, and dedicated pipettes.

Additional Observations:

• The same PCR protocol initially worked well for SFTPC-Cre, but has now become problematic with streaking issues that are consistent across different runs.
• Other genes, like p53, amplify correctly with the same DNA and reagents, indicating the issue might be specific to SFTPC-Cre and G12C.

Given that the primers and protocols are established and recommended by the standard lab, I am puzzled by the recurring streaking problem. Has anyone encountered similar issues with established protocols, or can suggest further troubleshooting steps to resolve this?

Thank you in advance for your help!