Week 4:
University of Iowa Department of Biochemistry

June 19, 2013

This week was a mixed bag of good and bad as far as the science goes. We injected 3 more constructs (H29, H31, and H32) which was awesome with a reasonable and functional number of tadpoles expressing our constructs.  Our earlier construct tadpoles are now huge and will be ready for analysis in week six assuming the expression of the constructs doesn’t disappear.  This has happened in the past for Yuan, but I’m hopeful it’ll be alright.

The not so great news was our H33 sequencing results showed what we had was not our construct, but rather a random strand of DNA that somehow managed to come out of our ligation.  This may have been due to a low concentration of H33-fragment 1, but we’re not entirely sure.  We’re working through a couple troubleshooting steps including running gels to check the size and purity of the fragment.  We’re also digesting the fragment in high fidelity NEB enzymes instead of mixing high fidelity with regular enzymes.  We want to digest the fragment with the same enzymes the vector is digested with so we can insert the fragment into the vector.  All NEB enzymes work in certain conditions like whether BSA is required, and what temperature will activate the enzyme.  These enzymes also have different functionality in one of four buffers.  We were using NotI-High Fidelity(HF) and AgeI in NEB buffer 4.  NotI-HF has 100% functionality in buffer 4, but  AgeI has only a 75% functionality rating in buffer 4.  On the other hand, AgeI-HF has a 100% functionality rating in buffer 4.  So we’re trying the digestion with NotI-HF and AgeI-HF in buffer 4 this time around.

My construct, H34 is also having some troubles.   I started the cloning process on Sunday and by Friday had determined that I needed to start over because I was convinced by that point that the H34 insert that I had created was not what I wanted it to be.  So starting in week 5, I will start the process again with Phusion PCR of my predicted H34 fragment.  Let’s Hope for the best.

Sarah was on vacation and Dr. Baker was at a conference this week and their absence was noticed.  It really didn’t have anything to do with the less-than-progressive state of affairs in my research, but the atmosphere of the lab’s social environment was a little less robust than usual.  Joe, Madestos, Yuan and I made up for this by going on a lab lunch trip on Thursday where I was introduced to the Pie Shake at the Hamburger Inn.

I would have taken a picture of the actual product, but I was just too excited and forgot about it completely.  They take a slice of pie (I chose a raspberry, apple, and rhubarb pie) and blend it into a milkshake.  Now I’m not one to miss a meal, but I didn’t eat dinner that night because I was still full from the lunch.  My ability to perform tasks in a normal state of mind after consuming the entire thing as well as a lamb pita sandwich from Oasis was… inhibited, so of course Joe and Madestos gave me a hard time about it.  All in good fun, of course.  But it didn’t stop there.  “Dave! Let’s go get a Pie Shake,” or “ You look like you could use a Pie Shake” have been thrown around more than a few times since the incident and I don’t think I will go anywhere by the end of my time here.  But it’s all in good spirits and reminds me of my own family dynamic, so it’s tolerable at the very worst.

The time I have spent here has really made me seriously consider a career in research.  It is a pleasure to work with the member of Dr. Baker’s lab, and it’s great to be able to listen to their conversations and be able to contribute even a little input that may be helpful.  My ability to actively participate in these conversations and contribute to the lab is entirely dependent upon my experience at Cornell.  My desire to perform research in a laboratory will now fuel my work ethic at Cornell in a way that I did not have before this experience.  The future seems real.  I can actually imagine what working in a lab will be like because –and I know this is a stretch, but bear with me- I have had a little experience working in a lab. It’s hard to believe, I know, but it’s honestly invaluable experience that fills the gap that’s inevitably left in undergraduate classes.  However, class is not devoid of value and gave/gives me the background and theory required to participate in research.  It’s just the act of being in the environment where you participate in primary research gives a unique perspective I’ve only achieved through immersing myself in the environment.