Problem	Solution
Significant degradation is observed during TGT labeling	TGT enzyme is contaminated with nucleases; test for RNAses and subject enzyme to two rounds of Ni-NTA purification. Alternatively, the concentration of RNAse inhibitor in the labeling reaction can be increased
Low labeling efficiency is observed	Validate TGT enzyme and preQ1 derivative using “Procedure for validation ofTGT enzyme and preQ1 derivatives,” described above. Labeling of ECY-A1 should be >90%. Re-express TGT if necessary, minimizing storage time at 4 °C or −20 °C, and storing purified aliquots at −80 °C. Check purity of preQ1 derivative; certain derivatives may be less well accepted by TGT
	Reaction can be optimized by increasing TGT concentration, preQ1 derivative concentration, or length of incubation. If a negative control is desired, a G → C mutation to the TGT recognition element can be made (Alexander et al., 2015)