I found 2 interesting articles for the detection of bacteria at Phylum level with MGB probes, claiming that they work.
I want to use that primers however when I use the oligoanalyzer tool, it shows that the Tm of the primers es 6-8°C higher than the probe. and in the other set of primers there is high chance of hetero-dimer. Delta G: -15.03 Kcal/mole
I am a bit skeptical that they work, however, I need them, but I do not want to make an expense that is not worth it.
This is one of the articles: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742902/#pone.0007125-Armougom1
I already know that in theory they shouldnt work efficiently but I want to know if someone have experience with this Kind of conditions?
I think it works and because of lower Tm they used MGB.
But I will glad to hear from others.
Regards.
MGB probes can be up to 10 degrees higher than calcelated Tm for native oligonucleotide
Agreeing with Andrei, the temperatures of all primers have a range of proper annealing. I have experience with plus or minus 10 degrees from ideal annealing temperature. When in doubt set up a PCR titration using the same reagents but changing the annealing temperature in your PCR conditions. Then run all samples on an agarose gel to compare the quality.
Depends on your PCR conditions, sometimes it works but not always. I have tried and worked it for me. Even if your probe sequences have less Tm than primers, the MGB at 3' end increases the Tm and stabilizes the probe.
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