Long-Overdue Update

by Lorien E. Menhennett

Well, it seems it’s been 2 months since I last posted. How time flies, doesn’t it? But I’ve been keeping myself out of trouble, I promise. Here is a bit about what I’ve been up to …

I wrote in a previous post that I had a tentative part-time job at the University of Illinois-Chicago Anesthesiology lab where I worked as an unpaid assistant for two summers (2010 and 2011). I started that job (paid this time!) back in mid-August. Working there, being paid to do what I do, and having so many more responsibilities than I ever did before, I feel just a little bit more like a “scientist.” If that makes sense. I’ll see if I can explain.

When I was a summer lab assistant at UIC, I worked with a wonderful post-doc named Olga who has become something of a mentor to me. She still works in the same department, and her lab room is actually next to mine now. When I have a question about something, I usually go ask Olga. Not only will she help me (or help me figure out where to find the answer to my question, if she doesn’t know), but she does so happily and willingly. Not everyone is like that. Olga taught me to pipette, to set up PCR, to run gels, to culture cells. It was while I worked with her that I discovered my love of bench science and research.

With the wonderful background Olga provided me, as well as what I learned in my post-bac coursework and research at Dominican, I felt I was ready for a position with more responsibility and autonomy. Well, I got it! While there was a bit of a rough start, things are going quite well now. And in that rough start, I learned a great deal about the scientific process, and my aptitude for it.

I was hired at UIC to genotype mice. My department is trying to breed double knockouts (DKOs, for short) of several genes in order to study these genes’ combinatorial roles in lung diseases. Genotyping mice, at least the way we are doing it, involves (in part) two techniques that are quite familiar to me: PCR and gel electrophoresis. I did both of these with Olga, as well as in my Research Methods course at Dominican. But genotyping involves many more steps as well, steps which were new to me. In addition, while I had done both PCR and gels, I had never done them completely from start to finish. By that, I mean from ordering primers to taking the gel photo in the dark room. I had done the middle part – the actual PCR and the gel – but not the initial and final steps. So I definitely had a lot to learn when I started this new position.

So here is the basic outline of the genotyping process:

1. Cut small pieces (about 0.5 cm) of mice tails
2. Digest mice tails to extract DNA
3. Run two rounds of competitive PCR (one for each gene) to amplify the DNA
4. Run a gel to determine whether each mouse is a wild type, knockout, or heterozygote for each gene
5. Analyze results, and hopefully set up new breeding pairs if you get any DKOs

Steps 1 and 2 were brand new to me. But I am proud to say that I am now a mouse anesthesiologist and surgeon! On one of my first days at the lab, my supervisor took me down to the animal facility and taught me how to anesthetize the mice, tag their ears, and snip of a bit of tail into an eppendorf tube (being careful to wipe with ETOH in between each mouse to avoid DNA contamination).

One of my gels, from last week. Pardon  the over-exposure and the line of dNTPs  at the bottom. I'm still working out the kinks with my primer concentrations and still getting the hang of working the darkroom camera ... it's a work in progress, but I'm getting there!

One of my gels, from last week. Pardon the over-exposure and the line of dNTPs at the bottom. I’m still working out the kinks with my primer concentrations and still getting the hang of working the darkroom camera … it’s a work in progress, but I’m getting there!

The tail digestion step was a bit of a debacle to begin with. I started out with a complex and time-intensive protocol that was handed down to me by another post-doc at the lab. It involved using proteinase K and several other reagents, heating the tail tubes in a 55°C water bath for four hours, and then inactivating the proteinase at 95°C for 10 minutes. When you counted prep time (thawing reagents, pipetting, labeling tubes, etc.), the whole process lasted somewhere between five and six hours. And I was getting very inconsistent results. But honestly, I wasn’t sure whether it was the tail digestion or the PCR that was the problem. For example, my primers could have been bad, or I could have been over- (or under-) digesting the tails. I literally worked with this protocol for a month, to no avail. Then my supervisor suggested I go talk to the woman, Debbie, who runs the Molecular Core, where the PCR machines are. I asked her how long she incubated her tails for.

“Oh, I use a kit,” she said, nonchalantly. “Why, what are you doing?” After I gave a brief outline of my method, her co-worker literally busted out laughing and said, “Wow, you’re really doing it old school!”

This “kit” is a miracle: while my protocol was taking five to six hours, the kit takes 30 to 45 minutes. And, as if that weren’t enough, the kit comes with a pre-made PCR master mix that contains a Taq JumpStart antibody which prevents the Taq from activating at room temperature … meaning you can set up PCR on the lab bench rather than on ice! It’s absolutely amazing.

Debbie let me borrow her kit to try it out, and it worked beautifully. My results are now consistent, and I even determined last week that we indeed did have several DKO mice – three males and one female.

While it’s obviously exciting to get results, what’s also exciting to me is the process of science. I faced frustration, and I didn’t give up – I worked on figuring out what was going wrong. Also, one of the things that always amazed me about Olga (when I worked with her those first two summers) was that she always had several experiments running simultaneously, and somehow the timing all worked out. I am learning how to do that as well – how to time my agarose melting, reagent thawing, and PCR and gel running (along with meticulous notebook note-taking) so that I get the most out of my time there.

I work at the UIC lab three days a week. This past Thursday I was working from home on freelance writing. When I woke up that morning, I felt a little sad, and thought to myself, “I wish I were going to the lab today.”

I think that’s a good sign.