Since last blog post, things have been making progress.
Since the primers obtained from Aday et al. have high tendency to self and hetero-dimerize, I got to design my first pair of primers ever and they worked perfectly well with Advantage Polymerase for the Histone-Gapdh control. I also optimize the IP protocol so we got three times as much DNA yield as before, which is a great success. I got to start the main part of my project-Ahr-Nrf interaction right away! It was very kind of Larissa to dose the embryos with TCDD for me because she does not want undergraduates to work with the toxic dioxin. TCDD activates Ahr to translocate to the nucleus where it binds to Arnt and induce transcription. Different from histone-gapdh direct binding, Ahr has to dimerize with Arnt before interacting with DNA, therefore there are still lots of troubleshooting to do to stabilize Ahr-Arnt-DNA crosslinking. The protocol for DMA (Dimethyl adipimidate dihydrochloride), a crosslinking reagent that reinforces Ahr-Arnt interaction, varies from paper to paper, so I am still spending sometimes figuring out the best concentration and incubation time for my ChIP project. I am also starting to use all the XREs primers designed by alumni James Meyo since last summer.
The weather is getting warmer this week. It has been 7 months since I started this project and we are only a few weeks away from finishing up thesis. March is indeed a crazy month, since it is the only month throughout the entire academic year that we don’t have any break. It is also the month for lots of job/internship/fellowship applications so everyone is very busy finishing up the semester as well as taking care of future/summer plans. Members in our lab are also doing well with job application. Some of us already gotten tentative job offers and some others got call-backs for interviews. We indeed have learned and acquired a lot of skills during the past 7 months in lab, which definitely well prepared us for the job market. Though it is not the end of the semester yet, I am thankful for all the opportunities I have had in this lab, and all the help I have received from Larissa and my labmates Nick, Nabil, Kathleen, and Sophie.
Until next time!!!