I am currently optimizing a RPA-based protocol. As everyone knows, this nucleic acid amplification technique is based on isothermal amplification (around 37-42˚C) in combination with recombinases and single-stranded DNA binding (SSB) proteins.
I have designed several candidate primers to optimize my RPA. All of them were searched in literature and designed considering the criteria to be used in an RPA reaction.
These same primers were verified by conventional PCR (At different Tm: 55-65ºC), obtaining a successful result.
The problem started when I used the RPA master mix (lyo version from Twistdx) and performed the RPA reaction with the same primers and samples used in the conventional PCR. ALL THE RESULTS BECAME NEGATIVE?!
Primer concentrations used in RPA reaction was 400nm (recommended by twistdx) and the reaction time was 30 minutes at 39 °C (conditions recommended for the set of primers tested). Visualization of the amplification was done on agarose gel (1.5%) and doing a posterior melting curve assay. In all cases, no amplification was detected.
Does any one have a clue on what is happening???
I don´t know which other variables I can change to obtain good results
Suggestions?
Thank you
It will be classic but you mentioned using a 30-minute reaction time, consider extending it slightly, as sometimes longer incubation times can improve RPA results, especially when using DNA extracted from complex samples. While 400 nM is a typical primer concentration for RPA, you can try titrating the primer concentration to optimize the reaction. Sometimes, higher or lower concentrations may work better depending on your specific setup. While the primers worked well in PCR, they might not be optimized for RPA. You could consider redesigning the primers specifically for RPA conditions. RPA primers may have different requirements, such as longer lengths (usually 30-35 bases) and specific GC content. First things that come to my mind. Good luck.
Primer concentration is important in this experiment (usually in all amplification experiments). In addition to it,my question is whether the recombinases enzyme and sample in your experiment are working properly?
All the best
Sara Melo-Dias
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line