Dear all,
I am detecting mRNAs with hairpin molecular beacon. The 5' end of the hairpin oligo is modified with BHQ1 and the 3' end FAM. After I dissolved the hairpin in NF water, I dispensed one drop on a glass substrate and checked the fluorescence under microscope. The molecular concentration is 10 uM. The fluorescence was super strong and the light really was really scary.
Does anyone know why the quencher cannot fully quench the FAM?
Thanks a lot for any hint!
Binbin Cui
Hi Binbin,
Quenching is practically never 100% efficient. BHQ-s quench by FRET, as far as I know, and this is distance dependent. If the concentration of the beacon, and of the fluorophore, is indeed 10 uM (which is huge), even a relatively tiny fraction of the original, unquenched fluorescence (which remains after quenching) appears to be bright. I would try a 100-times lower concentration of the beacon in the presence and absence of its target, and compare the fluorescence under the two conditions.
Regards,
Peter
Hi Binbin,
Quenching is practically never 100% efficient. BHQ-s quench by FRET, as far as I know, and this is distance dependent. If the concentration of the beacon, and of the fluorophore, is indeed 10 uM (which is huge), even a relatively tiny fraction of the original, unquenched fluorescence (which remains after quenching) appears to be bright. I would try a 100-times lower concentration of the beacon in the presence and absence of its target, and compare the fluorescence under the two conditions.
Regards,
Peter
Also consider the solvent. If you dissolved it in pure water, there may be enough backbone charge repulsion to prevent the molecule from folding up correctly. If the pH is alkaline, the RNA backbone will degrade. Put in some buffer at neutral pH.
Fam needs slightly basic for fully fluorescence, 10uM is way too high in concentration.
Dear Peter, Adam and Guobing,
Thank you all very much for giving me the precious advise. I will dilute the hairpin and try it again!
Best regards
Binbin
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