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!
Keep up the good work with your project! I hope that your powerpoint presentation preparation is going well.
ReplyDeleteHey Grady! Congratulations on Stanford, by the way. I understand what you mean when you say that you feel like your work is beginning to be rushed, especially with the end of our internships coming up in two weeks. My own work has become a lot more pressurized as I try to finish large amounts of research and incorporate it into my presentation. I hope the rest of your project goes well, and I can't wait to see how it finishes out!
ReplyDeleteHey Grady! Thanks for sharing some of the problems you were facing and how you dealt with them. That really helped understand what kinds of possible error there could be with your project. Nonetheless, another excellent post and best of luck as you enter the final stretch! :)
ReplyDeleteHey Grady, too bad the caps for the reaction tubes weren't completely sealed, but hey, those things happen, just glad that they weren't too expensive. I hope I can see next week how the reactions will turn out. Good luck!
ReplyDeleteHello Grady! It's unfortunate that time is starting to become a problem for you, and that your work is starting to become more rushed. It is nice to see that you are still learning from the process though, no matter what difficulties you encounter. I'm still looking forward to the next two weeks, even though the time is starting to wind down.
ReplyDeleteHi Grady! I'm sorry that you're feeling pressured by the time constraints on your project - it must be really stressful trying to get the maximum results in a short amount of time while maintaining experimental quality. It's great to hear that you got a clean sequencing result despite all this pressure! I'm also inspired by your determined and unwavering attitude towards the mistakes you make, and that learning from them motivates you to improve your experimental process even more in the future. I look forward to seeing how your project finishes up, and wish you the best of luck!
ReplyDeleteHi Grady! I am really sorry to hear that part of your results got ruined. Though, it is good to know that the mistake did not cause too much damage. I wish your the best of luck on the rest of your project!
ReplyDeleteHey Grady! I'm sorry to hear that you're starting to feel rushed. I hope that you're able to come back from the bad results. Good luck!!
ReplyDeleteMistakes come with the process. I'm glad you made it a learning experience. I wish you success on your future labs. Good luck and you got this.
ReplyDeleteHi Grady! Glad you learned a lesson, and I am especially glad that it was not an expensive one. I know its crunch time, so good luck getting those results!
ReplyDeleteHey Grady, good luck getting your project done. School can be stressful, especially BASIS, but I'm looking forward to seeing the end results :)
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