I'm new here and quite happy that I found this place. Maybe somebody can help me...
In mmy research project, I'm doing mostly DNA solid Phase synthesis (phosphoramidite method) and I am trying to establish an assay.
I have a double strand - at one 3'end I attach a fluorescence quencher.
The complimentary strand has a fluorophore at the 5'end.
So usually one would use a commercially available simple 5'-amino modifier and attach the dye via NHS-ester.
This is not working in my case, since There are additional modifications at the 5'end - so the dye is not really the very last. So I ordered this Amidite here and used it as the Last Base prior to the Position of the fluorophor:
Amino-Modifier C6-dC-CE Phosphoramidite from Linktech
I was optimistic that it would work - that the fluorophore would be fully quenched, when the quencher-strand is present... I mean, if I look at it 3-dimentionally, the quencher would literally stand rather downside, while the here attached fluorophore is more vertical... but anyway - I should see at least a some quenching, since the linkers are long enough and flexible (Not too long, though, as I do know from a positive control)...
BUT: It's not quenched at all
I am not getting it. Can anyone think of a reason? is There another option to add an amino-modifier internally - I Know one from GlenResearch, which would not couple in my hands... Do I oversee anything??? I really should see some quenching, even if the conformations of dye and quencher are Not fully optimal