Tag Archives: RF2016-Week6

Measure Twice, Cut Once

With two weeks left until the poster session, my project is finally starting to come together. I will definitely be working overtime to make sure that everything is completed in time.

As far as my project goes, I’ve improved quite significantly at using MATLAB, so things are going much more smoothly. I still find that, due to my inexperience, I lack necessary foresight to avoid making mistakes. At times, it feels like after taking two steps forward, I take one step back. Although this can be frustrating, it truly is the best way to become familiar with the programming language, and the field itself. This foresight only comes with experience, as evidenced by the progress I’ve made in just six weeks.

In addition to working with MATLAB, for the past few weeks I have been reading through neurobiology and neuroengineering textbooks, to obtain a deeper understanding of the evolution and fundamentals of the field. It’s interesting to see how improvements in technology have paralleled our ability to record activity from the brain, exemplifying the idea that many fields of science inform and develop each other.

Unexpected Outcomes

When I first saw my research project for the summer and what I would need to do to accomplish it, I thought my tasks were pretty straightforward and I would be done in two weeks with clear results. Well, turns out I was somewhat naïve and uninformed about this whole doing science because that didn’t turn out to be the case at all. I was unaware of unexpected outcomes. We finished the sixth week now and I just hope that I’ll be able to get my results by the poster presentation session which is in two weeks.

My project involves mutation of a particular gene using PCR and running Western Blots on human cells after transfecting them with these mutated genes as I mentioned multiple times in my previous blogs. I need to go through this process multiple times for different regions targeted for mutations and compare the results. So far, I have been able to get my mutant genes but it took 5 weeks for me to do it which would normally take one if everything went smoothly. Of course, nothing went smoothly. I encountered problems and made mistakes at every step of the way. Sometimes, PCR didn’t work, sometimes transformation and sometimes the transformed plasmids didn’t have the desired plasmids. I had to optimize the method by adjusting the temperature, tweaking protocols and adding some different chemicals to make the experiments work. Eventually, I was able to get the mutations I wanted, and the feeling of overcoming problems and reaching my goal was quite pleasing.

Now that I finally have my mutations, I will proceed to the next steps which should take about four days –again— if everything goes smoothly. I hope that I can troubleshoot my way into the results before two weeks. Although the problems I had to cope with and the mistakes I made were occasionally frustrating, they taught me one valuable lesson about research as Thomas Edison said: “Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time.”

Week 6: The Joys and Woes of Research

The first part of my research experience went surprisingly smooth, with few mishaps or questionable results, with the exception of an enzyme or two not working as well as we’d hope. It was fantastic to see my first plasmid I worked with (expressing GP120 and Red Fluorescent Protein) successfully be integrated into the poxvirus genome; some cells in our tissue culture glowed red, confirming the gene’s uptake!

The second plasmid I’ve been working with has required much more trouble-shooting, however. After several days of digesting and ligating pieces of DNA, we finally grew up a culture of E. Coli that we’d transformed with our plasmid (to make much more of it to work with). At first glance on a gel its size looked right. But after doing digests to confirm the genes we desired were present, we quickly realized something had gone wrong. After a few attempts to isolate the plasmid of interest from the overwhelming amount of ‘wrong’ plasmid, we finally decided to take another route. We’d order a synthesized segment of the plasmid to work with, making it more straightforward of a process.

While this setback wasn’t major or devastating, it showed me how valuable troubleshooting and the ability to adapt is to research. Things can quickly take an unexpected turn, and it is good to be prepared to deal with those issues and find a way around them. All in all, I’ve extremely enjoyed my research experience thus far and am looking forward to continuing work on this project!

Data, Data, and More Data

As the summer is getting nearer to its completion, so is my project, and fortunately with usable data! After four weeks of counting germination proportions in 1000+ petri dishes with around 20 seeds each, it’s a relief to know that each plate was actually accounted for and that the excel sheet is exactly the size it’s supposed to be. From what I’ve seen so far, there are some pretty interesting trends in the data even if not entirely what I expected (though the results will be more obvious once I finally figure out how to use R). Though by the time the poster session arrives, I’ll have graphs ready that can better illustrate the interaction between light quality and Flowering Locus C expression and their impact on germination!

Fortunately, there’s not much you can do to seeds that they won’t manage to survive or recover from. Nonetheless, I think I now have the lab record for the highest number of plates dropped during seeding (which involves placing the seeds onto the agar in each petri dish). I somehow even managed to fling a petri dish across the room using only parafilm (luckily, I had prepared 50 extra plates with agar, because I had zero confidence in my ability to not be clumsy). However, we had our last census this week, meaning that all of the plates were tossed in the autoclave bin to be subject to intense heat that kills cells so that the altered genotypes don’t mix with natural populations. After spending six weeks preparing the plates, seeding them, and counting germination proportions over and over and over again, it was a bit of a relief. I’ll definitely be much more satisfied by the complete graphs though, since I’ll know that all of the work yielded actual results!

Until then, there’s still a bit more work to be done!

Journey to Create RNA Probes

Over the past 6 weeks, I’ve learned a lot. I’ve learned how to pipette the optimal mix of reagents to amplify DNA for PCR. I’ve learned how to clone DNA with a plasmid vector. I’ve learned how to perform ventricle resection on zebrafish. And most importantly, I’ve gained a better idea of what research really is.

Research definitely takes time. Whether it was waiting a day for my bacteria inoculation or 6 hours for my RNA transcription, research actually involves lots of just waiting time. Thankfully, I was able to turn the waiting time into productive work by performing other steps or reading papers. Research also involves a lot of repetition. I can’t even count how many times I ran a PCR and gel this summer. It involves lots of repetition in the sense that repeating procedures makes you better at it. But also, troubleshooting. Sometimes, a step may not work and you may be stuck on one part for a long time, trying to figure out what’s wrong. This involves repeating the step many times and changing one thing at a time, trying all possibilities to get it perfect. I actually enjoyed this troubleshooting process because it was like solving a puzzle to me.

The main part of my project was to create 4 RNA probes for my genes of interest to perform in situ hybridization. This may sound simple to do, just create four strands of complementary RNA. Easy, right? The reality was far from this expected simplicity. In fact, it took over 6 weeks to create three probes (one gene refuses to be amplified and after tons of troubleshooting will still not work) that I just finished this Tuesday. This is really exciting for me because I can finally run all my in situs with my probes. This week will be the week of results where I can see where in whole 3 days post fertilization embryos, 6 week zebrafish hearts, uninjured zebrafish adult hearts, and injured zebrafish adult hearts the genes are being expressed. I cant wait to complete these final stages for my in situs.

In the journey to create RNA probes, I got to learn and experience a multitude of diverse procedures and techniques. Now, I feel much more comfortable performing these protocols and feel that I have a comprehensive overview of the common protocols related to zebrafish. Just the journey and learning everything both conceptually and mechanically have been so rewarding. At this point, results don’t even matter. This experience has been much more valuable (of course, it wouldn’t hurt to get some results).

Slowly by surely, I’ve been putting all the little puzzle pieces together. The past 6 weeks have gone by so quickly, I can hardly believe that the program is drawing near its end. It’s been such an enriching and thrilling experience working in the Poss Lab and I can’t wait to see the final results of my project!