FAQ: Some guides do not seem to be working as well as others with TtAgo. What can be done?

While we do observe that some guide sequences are more optimally utilized by TtAgo than others (please refer to GUIDElines for optimization of Tth Argonaute (TtAgo) reactions for detailed information and recommendations for designing successful guides), another potential source of problems can arise from incomplete 5’-phosphorylation of the guide. We have noticed that when this modification is ordered from synthetic oligonucleotide vendors, the level of phosphorylation and corresponding activity can vary greatly. To remedy this issue, we suggest one of two options: 1) order unmodified guides and phosphorylate the guides using NEB T4 Polynucleotide Kinase and the Protocol for creating 5′-Phosphorylated Guides from Unmodified Oligonucleotides for use with TtAgo or 2) if phosphorylated guides have already been ordered, simply carry out the same protocol in option 1 with NEB T4 Polynucleotide Kinase as a phosphorylation polishing step. In general, guides ordered with phosphorylation modifications perform well, however it should be noted that we observe guide oligos phosphorylated with NEB T4 Polynucleotide Kinase to perform as well or better than those ordered with a phosphorylation modification.