When I use RPA (fpg kit), I see a lot of primer noise on a gel. I need a clear band for a melting curve analysis. I tested different combinations of primers with litte result. If you have any suggestions on how to avoid this, please let me know.
Maybe you could try a primer modification with SAMRS (Self-Avoid Molecular Recognization System) bases. Nidhi Sharma‘s study shows that the dimers and hybridization between the primers could bearly form in RPA amplification.
Thanks for your comment. I agree that SAMRS could be a solution. However, the price of these modified primers in combination with the many primer pairs we wanted to test made us discontinue the project.
Yeah, SAMRS primers do cost a lot. The primers I used in my current assay (TwistAmp Basic Liquid) was designed by Primer-Blast with some parameter modification, however, primer dimers still appeared.
Recently tech support of Twist Co. provided a primer design software for RPA primer design, named "PrimedRPA". I'm thinking if this could solve the dimer problem. While this need a python backgroud......we do not have the technician. If you do, maybe you could try to design a few primers to run a test.
The primer dimer is also a big problem for our team, if you have the method to improve this situation, would you like to share it?
Thank you!
I would not mind sharing but I have moved on to other projects. Good luck with your work.
Thanks you. I'll try some time for this dimer. If we found the ways to improve this situation, we will let you know. And good luck with your research, too.
It is impossible. We have discussed the problem here https://www.biorxiv.org/content/10.1101/2020.03.02.969055v1.full
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