Non-specific amplification at 2nd PCR
If non-specific amplification is confirmed by 2nd PCR and 2nd PCR product, in may cases, there could be the problems at 1st PCR.
Please check difference of Tm values between forward primer and reverse primer with our 1st primer designe tool which you can download from download section.
Also, most troubles can be solved by using the high-fidelity PCR enzyme such as PCR enzyme in BCF4050.
If you are using PSS4050, we recommend a PCR enzyme that supports two-step cycles such as Toyobo’s KOD Plus Neo.