- Even a small percentage of probe molecules that are missing the quencher group can significantly increase background fluorescence and can interfere with an assay. IDT therefore strongly recommends that all oligos made with dark quenchers be purified by HPLC.
- BHQ-1TM and BHQ-2TM are available from IDT as 3′-end modifications which are attached during oligo synthesis using BHQ-CPG. QSY-7TM is attached to oligos using NHS-ester chemistry after synthesis and can be positioned at the 5′-end, 3′-end, or at an internal amino-dT base. IDT recommends use of BHQs for 3′-end modification and QSY-7 when 5′-end or internal modification is needed.
- Dabcyl should only be used when the reporter and quencher will be in close physical proximity, such as Molecular Beacons.
- Black Hole QuenchersTM produce an extra peak if examined using MALDI-TOF Mass Spectroscopy at 300 daltons below the true molecular weight that results from dye cleavage during laser desorption. This peak is an artifact and is not a concern. For more information about Mass Spectroscopy analysis of oligonucleotides, click here.