Worst day in lab

Wednesday evening, we showed our poster to the lab members for constructive criticism and some proof reading. I had the least data and prof suggested 2 new experiments for me to do which I immediately did the transfection for that night. Today I woke up especially early so I could get a ride from my mum to school. I reached lab at 8 and started my cyclohexamide treatment, it was my first time doing it and I had 3 sets of 6 samples each. Then I cleaned and set up 4 gel plates during the 1 hour break to cast the gels I wanted to use later. Also added NaOH to the EGTA solution which wouldn't dissolve since yesterday night. One of the gels leaked and in between my cyclohexamide treatment I made another which still leaked. I harvested the 18 samples without much trouble, though it took quite long. Jsenior informed me that there was probably not enough cells after her experiments so I won't get to do a forward transfection to repeat my experiments. It wasn't anyone's fault but I was quite upset :( Close to 11am, I started harvesting my other sets of cells to do a nuclear and cytoplasm extraction, also a first for me. Finding the right person for the protocol yesterday took me so long and I was silly enough to insist on making my own buffer -- that got me home at close to 9pm. Today shall be better, I thought. I harvested the cells and followed the protocol until it was the 15 minute incubation on ice. I started to prepare 10% NP40 from the 100% and it just wouldn't mix properly with water. It clogged up my pipette and formed lots of bubbles when I tried to eject that disgusting blob of NP40. This repeated twice and I was on my third try when the NP40 just stuck to the outside of my tip and refused to budge. I was impatient and panicking because 15 minutes was up and I didn't want my cells to swell too much. I was asking FYP mate A for help, we discussed abit then I spotted FYP mate B and asked her as well. Then a FYP student from the other lab came over to kaypoh as she was walking by and started asking FYP mate A trivial stuff while I was in between them trying to figure things out.

Me aka overly attached fyp mate : Stop! I was asking her for help, don't talk now!

Her sounding a little offended: why don't you ask Jsenior?

Since J senior happened to be just around the area I asked for her advice too. Everyone was just telling me the same thing regarding the dissolving blablabla. FYP mate B and Jsenior were there counting the concentration and stuff. She asked to see the protocol which I didn't have printed out because my laptop can never connect to the printer. FYP mate A offered to print it for me and I could barely speak to tell her that the protocol was in my email, not my thumb drive because my eyes were full of tears and I was about to cry. I couldn't hold it in and started sobbing a little.

I didn't want to start new experiments this late in the FYP, within a hour of knowing the experiment, not as properly guided as I would have liked and well I was just hungry and stressed out and frustrated at the NP40. I knew I was going to miss lunch for the third day in a row and staying ridiculously late and I was so damn hungry having not eaten anything since 7am and being on my feet the whole time.

I took off my lab coat and went to get my matric card to go to the toilet but I ended up just sitting at my table to cry. Wah damn emo. I recovered, though not completely because tears started flowing again after they went for lunch and I was there washing my nuclear pellet. I had missed lunch so often and this honestly wasn't what I expected at this point of the project. It wasn't that I didn't work hard in lab, I worked so hard but got no data regardless or missed lunches or not. It just wasn't worth mistreating my body for. This whole fyp just isn't worth it. I don't want to do research. 

Before they went for lunch my sweet fyp mates managed to dissolve the NP40 and printed out my protocol while I stayed at my table blowing my nose. They also kept checking back on me throughout the day :') 

Anyways I went 'screw this gels, I'm going to eat' and only prepared my 26 samples for loading after lunch. I ran 4 gels together but to my dismay the 2 extended gels didn't run due to the faulty equipment and my dyes and protein had been sitting there for so long. By the time it was done with running and transfer, the ladder was almost gone and the dye and maybe proteins had leaked across the lanes. I cut the membranes and labeled them before putting all for a super short block of 30 minutes which would end at 8.15pm. 

It was only when I put the membrane strips into the primary antibodies that I realised a few of my blots were missing, I was panicking again and spent a good 10 minutes looking in the different dustbins. I found them later in the transfer tank which meant I didn't even take them out after the transfer was done. I didn't bother blocking and just cut and dumped them into the antibody tubes. 

I left lab at 9 and finished dinner at 10pm, even later than yesterday.

This post isn't meant to fish for sympathies, this is hands down the toughest period of my fyp journey and i want to keep a detailed record. I hope to look back at the entry in the future and think that I've grown stronger since this day.