Week 2 in Toronto

Our second week in Toronto, we continued our work with N2A cells and learned more about BioID and Gateway cloning. On Friday we transformed the LR reactions (which put the entry clones into destination vectors) for STYX and F1. We then did Minipreps with the resulting bacterial colonies, did a digest with the plasmids, and ran the digests on a gel to double check the cloning products. Once we made sure the cloning worked, we did transfection and then treated with hygromycin. Since the vectors code for hygromycin resistance, any cells that integrated the plasmid will survive the hygromycin treatment. The media is then replaced every few days for two to three weeks, after which the surviving cell colonies are pooled. The resulting cell line has inducible expression of the incorporated gene of interest (in this case, STYX or F1) and can be used for mass spectrometry. We also observed BioID with one of the PhD student in the Gingras lab, Reuben. Next week we’ll learn more about BioID, affinity purification, and mass spectrometry.