I am currently optimizing an 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 designed considering the criteria to be used in an RPA reaction.
These same primers were verified by conventional PCR (At different Tm: 40-58ºC), obtaining a successful result.
The problem started when I used de RPA master mix, performing the reaction with the same primers and samples used in PCR conventional conditions, obtaining finally negative results. Primer concentrations used in RPA reaction was 400nm and the reaction time was 20 minutes at 40 °C. Visualization of amplification was done on agarose gel (1.5%) and also using an RPA-probe. In all cases, no amplification was detected.
After these results, I changed primers concentration (300-600nm), time reaction (20-30´) and temperature reaction (39-45ºC). Obtaining the same negative results.
I don´t know which other variables I can change to obtain good results
Suggestions?
how long are your primers? RPA works much better with long primers even though the annealing temperature is very low compared with pcr
That sounds ok. It might be worth titrating Mg concentration which is usually higher than in pcr. It may be that the Mg has a great effect on the primer binding.
- Badges
- Science method