I'm busy with validation of my primers nowadays. I have designed 5 primer-probes for Covid19 and when I check them by RT-qPCR as singleplex I don't have any problem. The oligos are synthesized by Macrogen. But then I do singleplex qPCR analysis to check RNASE P which is designed by CDC with the ROX probe. I got signals for NTC. Then, I order same sequence with HEX probe from another company but the problem was the same. Then, I check the NTC if it got contaminated in my device so I get some weak signals. Therefore, I thought there was a contamination in my NTC( nuclease-free water) But when I check, the water with another device they did not give any signals. What can be the reason for this weak signals? Is the problem probes or device?
I am not sure, but recently I designed a couple of primers to amplify a gene by qPCRs, I noticed a similar thing. At first, I also thought it was contamination, so I changed all my reagents. However, the problem was that my qPCRs produced primer dimers. I got rid of the amplification in the NTC until I used the third pair of primers. I also ran controls, including only one primer at the time, and another control without primers, to make sure the primers are not the problem.
Şeyma Nur Polat It looks like a primer dimer formation. You can get rid of this by either of the following:
1. if using two step PCR program, the annealing cum extension should be done at 65 C (instead of 60 C) for the desired time
2. Increase denaturation cycle time by 5-10 sec
3. (If 1 and 2 do not work) Along with 1 and 2, use 2.5% DMSO in the reaction mixture.
Remember to do it sequentially and not all variations to be done together.
check your raw data it looks like free dye/ linear signal increase. If you see only a linear increase this is chemistry/ labelling. You might order the probes double quenched this could help you here
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