Hello! I'm working for the first time with RPA. The primers were already tested with qPCR and showed great results. Now, for the RPA we are using the same primers and using the TwistDx kit for the RPA. I am not obtaining any bands, even on the positive control.. could you give me suggestions of what may be wrong? Thanks!
It is difficult to give you a precise answer. I think it is necessary to check the steps below to find the source of the malfunction :
- Double-check primer and template quality.
- Ensure reaction conditions (temperature, buffer, enzyme) are optimal.
- Test positive control with proper template and reagents.
- Ensure reagents are not expired or contaminated.
- Optimize reaction conditions (time, primer concentration).
- Verify detection method (gel electrophoresis, staining).
possibly over amplification of a reagent contaninated with previously amplified product. Run 3 no template control samples and see if they amplify
I noticed the original primers are for qPCR. Are you using the same template molecule (e.g. viral RNA, cDNA, etc.)?
The reason I ask is that qPCR primers often don't work for genomic DNA (assuming your organism is eukaryotic & the primer is across a splice site).
You're getting a lot of non-specific amplification. Try less input molecules and that might help
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