Save those samples!

I let out the biggest sigh of relief when I saw those three letters in that 1.5 mL eppendorf tube sitting in my box in the -20C freezer.

It has been a couple months since last posting due to the fact that a lot has been going on in my life personally and academically. I have just gotten a chance to sit down, today, with my PI to discuss the project that I am currently working on and all the results that I have gotten.

A time crunch is an understatement for the situation that my project is in. I have been working on this since February, and since the middle of March, my project has been going in circles. Finally, after somewhat conclusive NMR data and yet again unexpected SEC-MALS data, we (my PI and I) have come to the conclusion that quite possibly, something as minor as a His-tag is affecting my entire protein sample.

It would have helped to realize this sooner, but despite all the failures that I have seen in the past couple of months, it is good to know that all my experiments and tests have not been in vain. Though I am going back to step one by transforming my plasmid with my target sequence into E. coli for overexpression, at least I know that what I am about to work on will give me a shot at producing some results at the very least.

My first year at Northwestern is coming to an end and my time in lab has been nothing short of amazing. I have pushed myself to so many limits and cannot have asked for a better lab to join. The people I work with are so incredibly talented and though it often times makes me realize how little I have accomplished in my life and how much of a failure I seem compared against my fellow labmates, I use that to drive me to do what I need to do.

I am motivated and determined to be able to push my project forward. It really is satisfying to see results coming out, to be able to interpret them on your own, and then discuss with your PI in a two-way conversation regarding what the next steps are.

This year has been such a whirlwind but I can say that I am so happy and grateful for the grant that I have been given to allow me to continue my research here during the summer months.

For now, this is it. Bacteria cells await my care. Just know, saving samples is crucial when doing research! Save those samples!

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A Level of Comfort

With experience, in any field of work or study, comes comfort. I have been a part of this labe for just over 4 weeks now. When I started, I got to pipet samples over and over again into eppendorf tubes. I followed protocols with the postdoc I am shadowing for the quarter watching me like a hawk, ensuring I did everything properly. At the beginning, she even did all the calculations and conversions for me.

Now, though I still follow protocols (since if I didn’t I might be in some big trouble!), I have a lot more freedom. I am left to complete experiments that I start. My postdoc doesn’t need to be around in order for me to complete something. For example, like yesterday. I came by between class and work to load my samples into an agarose gel to run. After the gel ran for 20 minutes or so, I came back to lab from work in order to visualize it and check to see if we got a positive result the second time around. This entire time, from when I loaded the gels to after I finished visualizing them, my postdoc was not in the lab. She was doing her own things and I was left to just complete the experiment. I have to admit, at the beginning, I was a bit hesitant. I felt like fish out of water, but after a split second of this feeling, I didn’t let it overcome me. I started to just go about the experiment like I would have if she were here.

And today, just before she left for her lunch break, she checked in with me to make sure that I was comfortable with the protocol I needed to follow in order to extract the plasmids from the cells. I told her I was okay, that I will be able to do it on my own. And indeed I am okay. Because I got sample at the end of following the protocol.

The samples will be sent out for sequencing Monday. The My bacteria grew nicely and now have found a new home in 4 degree Celsius.

Quick Update 10.30.12

It has been a while since I have posted. Since I last posted a week ago, nothing much has changed. To be honest, this week felt like it went by extremely slowly. I had an exam last week along with a paper due and four or five quizzes (too many to keep track of). Aside from that all, research has just been getting more and more interesting; each day I learn something new and each day I see the results of what I am working on. This is truly a rewarding experience.

For example, just yesterday, I inserted a TEV plasmid into E. coli competent cells and after growing overnight in an incubator, there were colonies on my agar plate. As weird as it sounds, the colonies are really pretty. I don’t know what it is about the small things of this research — growing colonies, seeing bands on the gel, extracting plasmids — but I am absolutely in love with what I am doing. Sometimes, I wish I could just skip classes and run tests all the time. Sure, there were a few hiccups here and there during the first couple of weeks where the bacteria didn’t grow properly or the plasmids weren’t extract or inserted into the cells, but these are minor compared to the end goal. To know that I have been matched with this lab and to be able to truly see the words transform into the laboratory is more than I could ask for.

But for now, this is all I have to say. I am just going with the flow and taking everything one step at a time. Nothing in life can’t be overcome; I just have to keep a positive mentality and just keep pushing forth.

Seeing Results: A first successful experiment

I started a little over a week ago at my lab. I haven’t received my own project yet as I am shadowing a post-doc in the lab, learning the techniques that are employed when conducting actual research projects.

To start, I was asked to transvect three different plasmids into E. coli and then extract these plasmids from the bacteria, linearize the samples, and obtain the linearized DNA to save for future use.

My first attempt in transvecting these bacterial cells with the plasmids didn’t go as smoothly as planned; after regrowing the bacterial culture with the transvected cells a second time, the plasmids were successfully extracted from the cells and concentrated so that all I had in test tubes was plasmid DNA. With this circular DNA, I got to linearize them using restriction enzymes and then proceeded to perform PCR to allow the DNA to amplify. After PCR, the products were run on a gel to check and make sure that the samples indeed were linearized properly and that the bands were of the right length.

Then, when all this was done, I expected to start on a new project, to learn a new skill. But I was wrong. Yes, I learned a new skill but I was not given anything new to work on. Instead, the post-doc showed me how to, after imaging the gel, extract the DNA from the gel and proceed to obtain this linearized DNA only. Even though I used a pre-made kit in order to extract my DNA from the gel, it was fascinating to see how all this is done. I never thought that I could do anything with the gel and the DNA bands that are produced. In the past, after imaging, the gel was discarded and that was it. The DNA was never saved. But I have always wondered, how do labs get such large quantities of linearized DNA of a specific vector if the only way that a vector can be amplified is by transvecting bacteria? Well, I have my answer.

I love lab. I love learning, how hands-on the entire process is, and how everything that I have learned in my past classes are actually coming alive and are applicable. I am glad I chose the major I did. I absolutely love it. And I can’t wait to learn more as the year goes on.

[Sorry for the lack of coherency in some parts. It is a pretty stressful week with several quizzes, a midterm, and a midterm paper due for me. Hopefully, I will begin to write more after all this is over and write with more finesse and eloquence. Thanks for reading!]

Starting from simple tasks.

They told me that as an undergraduate, all I would do is wash their used dishes and pipette microliters of substance a thousand times over. They told me that I would not ever do any meaningful research and that professors despised undergraduates.

Whoever told me this, I didn’t want to believe them, but I did. Rumors, they were.

I am glad that I kept an open mind going into my junior year. By doing so, I was able to meet somebody who knew a professor who was looking for an undergraduate to research in his lab and it just so happend the work the lab is doing relates to my interests.

I have been in the lab for a week. I have gone in three times now. Each day I have done something different. I have prepared bacteria to be transvected with a plasmid and grow, I have extracted plasmids from the bacteria that were grown, and I have made broth in which bacteria can grow. Each task, as simple as it may seem, has been a learning experience. I have not had to wash any dishes. I have not had to pipette micro quantities a thousand times over. What I have done is followed protocols. I have followed instructions given to me and been left alone to complete them. The post-doctoral student I am working with this quarter has helped me each step of the way, explaining why things are the way they are and then leaving me to do what i need to to. Nobody watches me like a hawk. And yet I am entrusted with the most basic yet critical tasks for running future experiments.

WIthout the work that I am doing now and the techniques I am learning, I will not be able to perform any future projects. It is critical (at least for the research the lab is doing) that bacteria can grow in large quantities and that plasmids are available in large quantities to work with and manipulate for future studies.

Everything I am doing has meaning. Everything I am learning has value. Nothing is mundane, nothing is useless.

I am building the foundations of what will eventually lead to an independent project and maybe even something more. But for now, I am taking it slowly, taking it one step at a time, and soaking it all in.