Instead of a daily schedule, I have more of a three-day schedule – well, if I am lucky enough and nothing goes wrong, which doesn’t normally happen.
As I mentioned in my blog last week, I am searching for mutations on the gene of interest that can potentially lead to bipolar disorder, so my project is really a combination of bench work and computer work. On the first of the three days, I will start by aliquoting a new set of patient samples into strips PCR tubes just so that later on in my experiments, I will be able to take 8 samples at the same time with a multi-channel pipette. Then, I will be sitting (or rather, standing, because I feel more functioning when standing) by my bench and preparing for PCR reactions. This involves calculating how much of each reagent I need, actually mixing those reagents, and trying to do things as quickly and as accurate as possible because the tubes can’t stay out of the ice bucket for too long. Depending on the number of samples I have, how long this step takes varies. Last week, I did 96 PCRs at the same time (which I should probably never try again…) and it took me almost 3 hours merely setting things up. The actual PCR reaction takes place in a thermo cycler and takes about 2 hours. As I am waiting, I will be either reading papers and scribbling down notes, or helping out my mentor with his experiments. So far, I have helped him genotype mice and do Western blots. I really enjoy helping him because my personal project involves mostly repetitive work, and I always learn new skills when observing or doing his experiments. After my PCR finishes, I will take the tubes out and put them in the fridge, so that they stay fresh for the next day.
The second day is mostly gel electrophoresis. I do gel electrophoresis on each of my PCR reaction so than I can 1) see if the PCR actually worked and 2) determine if the PCR products are of good enough quality to be sent directly for sequencing. Unfortunately, my gels always disappoint me, because a lot of the times, although I do see my target band on the gel, there are also some nonspecific bands which really interfere with sequencing. I have been trying to optimize the PCR reaction by redesigning primers and adjusting the primer annealing temperatures to reduce nonspecific binding. The results are getting better, but problems still come up every once in a while. At the end of the day, after I finish running all my samples on a gel, I will send the clean and working ones to sequencing.
The third day usually starts early, because the sequencing company always sends me the results at 3am. So the first thing I wake up in the morning will be opening up my laptop and clicking on one of the chromatograms to check if the sequencing reactions worked. The rest of the day will involve mostly computational work. I will look carefully through each chromatogram, align the called sequence with the disease-free sequence from the online database and look for mutations in my patient samples. At the end of the day, I will document the mutations, if any, and the overall sequencing quality in my notebook. This will also be a day of discussion and Q&As with my mentor. We look through the chromatograms together and try to find out what we can improve on to make the sequencing results prettier. We seldom get perfect results, so there is always room for improvement. Figuring out what went wrong and how to fix it has been an enjoyable experience.
Ideally, I only need to do around 400 PCRs to finish my project. In reality though, I have done way more than 400 PCRs in the first half of the program. Things go wrong. Sometimes the PCR just won’t work – you can see how the polymerase had slipped off the template from the chromatogram. More often, I see mixed wave peaks in the chromatogram, which means that I have contamination in my PCR. I spent most of the time reading and asking about, and trying out ways to optimize PCR and to get rid of contamination. So although my project sounds simple, what I am learning in lab is not just how to do a PCR, but how to perfect my techniques by trial and error, how to fail again and again but stay resilient. I am also on my way of becoming an “expert” in looking at sequences, and the best way of accomplishing this is: well, I’ll just have to look at enough of them.
