Hi everyone!
I’ve spent lots of time in the last week playing around with the new downstream primers I ordered at the end of last week. I’m not 100% satisfied yet, but I’m very confident I’ll be there soon enough. I am starting to feel the time pressure of finishing in time for the presentation, and I think because of that some of my work has started to get slightly rushed if not sloppy.
In a lot of ways, what I’ve been doing is basically a repeat of what I already did with the first set of primers I’ve ordered. I’ve tried lots of different primer combinations and various reaction conditions, and it’s definitely helped significantly so far. The new primer set worked on one of the cell lines I’m working with right away, so I sent that product off for sequencing right away. An now, after just under a week of optimization, I think I’m getting a decently clean product on all 5 cell lines. I got an excellent read (~450 base pairs in length, which is near the upper limit for Sanger sequencing) on the sequencing for the product I sent out. That’s excellent news, because I now have more than enough of the rearrangement sequenced than what I need to design a qPCR assay. If I absolutely had to, I could do it with only ~75 base pairs on each side of the breakpoint, but it’ll be significantly easier with the ~200 base pairs on each side I have to work with now.
But because I’ve been trying to move forward as quickly as possible, I;ve started noticing I’m making some unnecessary mistakes. For example, I accidentally forgot to tighten the caps on all the reaction tubes before placing them in the thermocycler. That’s a big problem because PCR cycles alternate between ~60 to ~95ºC, meaning the water inside the PCR tube can evaporate if the caps aren’t on tight enough. When I took them out the next morning after the PCR cycles were finished, most of the reactions were completely ruined, now only a hard, green resin that’s totally useless. And because that was the nested reaction, it means I needed to repeat both the external and nested reactions in order to try that specific experiment again.
Luckily, thanks to the cheaper Taq polymerase I’ve been using, little mix-ups like these aren’t costing the lab in any significant way. I have learned an important lesson, however: for lab work, it’s always better to go slowly and carefully. Even if I get lucky and don’t make a mistake, having sloppy/incomplete notes for an experiment means that at best I’ll need to spend more time interpreting the results and at worst I’ll need to repeat it completely.
Going forward, I won’t stop trying to move forward with my project as quickly as possible, but I will be sure to do everything much more carefully.
Thanks for reading!
