What are the reasons for the low signal-to-background ratio after the synthesis and purification of a molecular beacon and how do I solve the problem?
Posted August 29, 2023
It is likely that the high background is a result of contamination by free fluorophores or oligonucleotides, which contain the fluorophore but not the quencher. Free fluorophores can be detached by passage through a Sephadex column. To ensure every molecule has a quencher, one should repeat the purification of oligonucleotides that are protected by a trityl moiety and labeled with DABCYL before coupling with the fluorophore. Another reason for the low signal-to-background ratio is the MB may fold into an alternate conformation which results in a subgroup that is not quenched efficiently. To solve this, one should change the stem and probe sequence. Another reason for the low signal-to-background ratio is the assay medium may contain inadequate salt and the stem opens up. To solve this problem, there should be 1 mM MgCl2 in the solution to ensure that stem hybrids are produced.