So I woke up at 8:30 am this morning and felt the need to go back to the lab to finish up what I was doing on Saturday.
I get off the bus at Eglinton. I forget my access card. This would be okay if it was a weekday but it’s the weekend. So I bus back home. Grab my access card, go back.
I get there. Did some calculations. Started to set up my westerns. Realized we were out of running buffer. The recipe on the container was messed and pretty much I had no idea how to make running buffer/I googled but didn’t know where the stuff was. Long story short, I figured it out eventually, but I couldn’t find masks anywhere and inhaled an amount of SDS worth a lifetime. Great. Legit took me an hour to figure out.
Moving on. I prepared my samples from my lysis on Saturday. Set up six westerns gels. Started loading my samples and the sample decides to float out of the well. This normally doesn’t happen.
So I did some troubleshooting. At first by myself.
My initial reaction was that my samples were too sticky because my boss doesn’t want me to centrifuge my samples. Normally you centrifuge your samples, because the stickiness is due to DNA and you can pellet that out by centrifugation. But I thought it was strange that my samples were still “sticky” because I upped my lysis buffer volume from 150uL to 230uL. The stickiness was resolved at 200uL but I still decided to up to volume just to be absolutely sure. So vortex my samples some more to mix. Still doesn’t work. I tried my other samples. They all floated out of the wells.
So I text my boss to see if I could run my samples through a syringe. See, you lose a lot by doing that, so I wasn’t sure if she wanted me to. She told me to add 50uL more of my sample buffer, reboil my samples, and that I shouldn’t do the syringe thing on my own. So I try to reboil my samples. (I only did 6 to see). The dry bath broke down right then and there. So then, I was on a mission to find another dry bath. After another good amount of time, it still didn’t work.
At this point, there was another summer student at the lab so I asked him. He “supervised” me while I ran one of my samples through a syringe. Annoying as shit process. Still didn’t work.
His girlfriend suggested I add 1:1 water to my sample buffer to see if that was the issue. Even the water mixture floated out. Hinting that my sample buffer was somehow contaminated. Then she suggested I reboil my samples and not put them on ice. Didn’t work.
At this point, I call my dad because it was already the afternoon, I had already spent a majority of my day there fucking troubleshooting, I was not about to leave empty handed. He did a good amount of yelling at me with regards to precision when it comes to lab work and hung up on me eventually.
Feeling like all 18 of my samples were about to go down the garbage chute (12 of which were my siRNA knockdown plus the protein concentrations were perfect this time around), I burst into tears.
I look up feeling like there was too much liquid on my gloves. My nose was bleeding uncontrollably. So I bleed out for a good 30 minutes in the bathroom and at this point I wanted to go home so badly. I was just done with life. But I realized I had everything still out I had to clean up and my nose still wouldn’t stop bleeding. And then I realized one of my samples was still boiling, and it had already been close to 40 minutes, I’m panicked. I legit run around the lab, still bleeding uncontrollably, clean up with one hand, and run back to the bathroom to try and stop myself from bleeding. Thank god it was a Sunday, the women’s bathroom looked like someone died in the sink.
I go home. Depressed. Sleep. Wake up. Looked up the composition of the sample buffer. Glycerol is the stuff that makes the samples heavy enough to sink into the wells. Went back to the lab at 10:00 pm. Put 50uL of glycerol in my samples.
And it worked.
So fucking stupid. Whoever decided to contaminate the sample buffer and not say anything.
(But while I was there, I found that two of my samples wouldn’t freeze meaning they must have been contaminated with ethanol or something of the sort.)