I incubate IRdye700 with antisense oligonucleotide phosphorotioate for about 12 hours in Na2HPO4 buffer. (MW IRDye700 = 1954; MW Oligonucleotide = 5844 )
When I purify the result using Aktaprime Liquid Chromatography with Superdex Peptide 10/300 GL column, we found that the oligonucleotide was not conjugated with dye.
Dear Dwi Heryanto, form the web page of the producer it seems to me that the IRDye 700 is selled only as phosphoramidite. It means that its incorporation must be done during the solid phase syntesis of the oligo.
Thank you Mr. Capobianco. But I use IRDye700DX NHS Ester and conjugate with 3'-amino modified oligonucleotides. We thought that NHS ester can react with amine on the oligonucleotide in post-synthetic labelling
OK Mr. Dwi Heryanto, in that case the procedure is correct. In my experience (but not with this dye) important issues are the pH (avoid the protonation of the amine but prevent the hydrolysis of the NHS ester) and keeping the concentration as high as possible. Consider also performing the reaction in water/DMF to better dissolve the dye if necessary. Moderate heating 40C can help. Do microreactions in eppendorf vials on a scale of 10-20 OD of oligo possibly with 40-200 microliters of solvent, then use HPLC to found the best combination of parameters. Good luck.
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