Starting Amino-Oligonucleotide requirements: The Antibody-Oligonucleotide All-in-One™ kit is designed to conjugate any high purity 5’ or 3’ amino-modified oligonucleotide (20-60 nucleotides in length) to any monoclonal or polyclonal IgG-class antibody. The protocol requires a minimum quantity of 10 OD260 and a maximum of 40 OD260 units of HPLC purified amino-oligonucleotide. Solulink recommends that longer oligo sequences (e.g., > 49-mer) be synthesized with a 5’-amino group and shorter oligos (< 49-mer) with a 3’-amino group if the specific application permits. Oligonucleotides <49 bases can be either reverse phase (RP) or ion exchange purified (IEX) while longer oligos (> 49-mer) can be IEX or double HPLC purified depending on the specific services offered by the vendor. Some vendors offer these purification options on a custom basis while others offer them as a standard service, albeit at additional cost. Be advised that unpurified 3’-amino oligos contain a significant quantity of truncated failure sequences that lead to undesirable conjugation products while unpurified 5’-amino oligos contain up to 50% of A260 units in the form of shorter unmodified failure sequences that never form conjugate and thereby alter the stoichiometry of the conjugation reaction. For best results always use the highest quality, HPLC purified amino-oligonucleotide available.
Note: Please be advised that some oligo vendors will not HPLC purify amino-modified oligos or in some cases longer oligonucleotide sequences (modified or unmodified) except as a custom service. However some oligo suppliers do offer these services as a standard option. TriLink recommends that customers always use HPLC purified amino-oligonucleotides in this protocol. We recommend requesting a mass spectrum to confirm the final quality when available. The mass spectrum confirms percent full-length purity as well as molecular weight (unambiguous confirmation of amino group). As a general rule, we do not recommend using crude oligonucleotide preparations to make a conjugate. Use barrier pipette tips and good laboratory practices at all times to avoid potential contamination and/or cross-talk between different oligonucleotide sequences.