MouseGenetics Forum > PCR & Genotyping Troubleshooting Thread: "Faint / nonspecific bands on new primer pair - annealing temp issue?" --- Post #1 by newgradstudent22 --- Designed a new primer pair for genotyping a floxed allele and I'm getting really faint bands plus a bunch of extra nonspecific smearing above and below my target band. I ran it at 58C annealing since that's what our lab usually defaults to.

The hack / solution

Alternatively touchdown PCR (starting a few degrees above your primers' Tm and stepping down over the first several cycles) works well too if you don't want to commit to one fixed temp, and tends to be pretty forgiving for suboptimal primer designs. Switched our SOP for this primer pair to 62C annealing and it's been consistent since.

📎Source: genotyping_annealing_temp_noise.txt

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