How can I prevent or reduce amplification in my no-template control (NTC) reactions from SYBR Green I assays?
Amplification in NTC reactions can either be from contamination or non-specific amplification. Performing melt curve analysis can help identify if the signal is from contamination or from non-specific amplification. If it is contamination, the melting curve of the NTC reaction will have the same Tm as your target sequence. Good aseptic technique, using aerosol-resistant pipette tips and a Real-Time PCR master mix with dUTP and UDG, can help to reduce any potential contamination. If the signal is due to non-specific amplification, the melting curve of the NTC reaction will have a different Tm than the target sequence. The most common type of non-specific amplification is primer-dimer formation, and there are a number of ways to reduce this. Optimal primer design is an important first step in preventing primer-dimer formation. • Primer pairs should be screened using oligonucleotide analysis tools for any secondary structure, self-annealing, or cross-annealing. • A hot-start DNA polymeras
