This revisited Thalassiosira pseudonana conjugation protocol builds on the following two protocols: Conjugation of Thalassiosira pseudonana (DOI: dx.doi.org/10.17504/protocols.io.f55bq86) & Pour plating of Thalassiosira pseudonana (Tp) (DOI: dx.doi.org/10.17504/protocols.io.jfncjme). Readers are strongly encouraged to read both before using this one. Gene-EGFP fusion depicted in the 'Expected Results' section was expressed under consitutive Tp-derived fcp promoter and terminator using pTpPuc3 episome backbone described in Karas et al. (2015).