Why do I need to use a cacodylate-containing buffer for the TdT step?
During the development of the TLAD method, the Roche TdT enzyme (from calf thymus) was originally used. However, in our hands the TdT enzyme did not perform consistently and varied lot to lot, which we speculated was due to this enzyme having been derived from a natural source (this seems to be tacitly acknowledged (our speculation) by the fact that they no longer offer the calf thymus-derived TdT and that they made this announcement (pdf)). The switch was made to NEB recombinant TdT (Roche hadn’t offered a recombinant TdT at that time; they do now). Curiously, however, the NEB TdT enzyme wouldn’t tail when prepared with the provided reaction buffer (NEB Buffer 4), and it was determined that this was due to the dTT in NEB Buffer 4 precipitating the cobalt chloride in the reaction solution. When the cacodylate-containing TdT buffer (provided with the Roche enzyme) was tried, however, the NEB TdT performed satisfactorily and consistently. Thus, we strongly recommend using the cacodylate-
