A dash of drama

I can say with almost absolute certainty that right now, my plate is full and my ability to deal with unexpected problems that are potentially disastrous is, well, lacking in grace. Blame it on hormones, lack of sleep, an over-ambitious work schedule, feeling like a fat blob...whatever. It just is.

I recently learned that one of the letters of recommendation required for my fellowship did not get uploaded onto the electronic form. I learned this after the deadline. The person responsible for this particular letter swears they turned in the letter. In fact, they are adamant that the letter got submitted before the due date and I believe them. I sent an email to my contact person at the granting agency explaining the situation, waited for a response and tried not to lose my shit.

On the one hand, I know my chances of getting the first fellowship I apply for is pretty slim, but putting that application together, while a great learning experience, was extremely time consuming and frustrating. I want it to at least get reviewed and not thrown out on some kind of technicality.

In the end, the association I applied to contacted my referent and ended up allowing them to send the letter of support through the mail. WHEW...

Talk about feeling relieved. I really expected the association to tell me they couldn't do anything since the deadline had passed. In fact, I was so sure of this response that I almost didn't even bother trying to rectifying the situation. Once I reminded myself how much work I put into the fellowship application, I decided that it couldn't hurt to try.

I cannot express how relieved I am that everything worked out and I am so thankful that the association I applied to allowed my referent to resubmit their letter of recommendation.

Cloning tard

That's me, at least for the last couple of days. Typically, I am a cloning rock star, but a combination of arrogance and laziness resulted in a 1 day process taking 3 days.

Here is the situation.

Currently, I'm in the midst of a protein trouble-shooting extravaganza and as a result my work load is significantly lighter than usual. To fill the gaps, I decided to pick up some cloning that I abandoned this summer.

2 days ago:

After much digging around in the freezer, I located the five abandoned PCR products, ligated them into a vector and transformed the ligation.

That's pretty standard. I left feeling confident that I would find five plates containing beautiful colonies the next morning.

1 day ago:

Success! The perfect amount of colonies were scattered about each plate. You know, not so many that you can 't pick a single colony, but not so few as too indicate that all you probably got back was a bunch of bullshit re-ligated vector.

Anyway, I colony PCR a few colonies off of each plate and run a gel, only to find that I didn't get a single PCR product! WTF? This doesn't happen...at least not to me. I stair at the computer screen in disbelief for a few seconds when I realize that in addition to a lack of positive results, I also lacked a single negative result. I should have one or the other.

I dig back into the recesses of my mind and vaguely remember that the inserts in this case would be pretty long. Most likely, I didn't use a long enough extension time.

I set up another colony PCR, this time with a longer extension. I run this PCR out on a gel and this time I see a couple of PCR products, but most of the gel is black. Seriously, WTF? A couple? What am I? A first year grad. student.

At this point, it's the end of the day and I really want to get the hell out of there, but I am not willing to admit defeat, so I decide to inoculate a couple of colonies from each plate.

Today:

I verify the long way (the short way being colony PCR the day after you transform). I mini-prep the overnight cultures, digest the plasmids and run a gel. The image of the digest comes up on the screen and it's all I can do not to yell out MOTHERFUCKING FUCK! It looks like all empty vector, except for the two that I already knew about from the day before. I walk down the hall in a huff toward my office.

I'm ready to trash three of the plates, but instead I decide that maybe, just maybe it's time I actually look at a gel from the summer and see exactly what damn size the PCR products I dug out of the freezer were in the first place.

Well, what do you fucking know? Three (that's right three) of the PCR products are exactly the same size as the vector I ligated them into. Damn it. The digestion was a complete waste of time.

I don't feel like digesting again with an additional enzyme because I don't know which one to use and at this point I am not in the mood to look it up (despite the fact that not looking shit up in the first place is exactly what got me into this mess). I decide once again on PCR, but this time I will actually use the correct extension time.

Conclusion, almost every vector contained the appropriate insert.

I could have found this out yesterday if I had just looked up the gel in the first fucking place and used the correct extension time for the first round of colony PCR. Now, I'm behind, I feel like an idiot and it's only Monday.

The ever-elusive carrot

My graduate advisor is a classic carrot-dangler. Unfortunately, it takes forever to get her carrots and when you do, you are so irritated and annoyed that you don't even care about the stupid carrot, instead you want to jump up and down on the carrot or shove the carrot into a dark orifice not located on your own body.

In this case, the carrot is a publication. I believe my graduate advisor means well, but two things typically go wrong on her part and one thing goes wrong on my part. For her, the two problems, usually, are (1) a complete inability to manage her time, ultimately leading to missed deadlines (we're talking months here not days) and (2) an unwillingness to learn from past mistakes and avoid them. My problem boils down to one thing; I continually fall for her claims that things will go differently, resulting in unnecessary frustration and sometimes, homicidal rage.

Let's take the current paper-writing efforts grad advisor and I are making. We started this paper in May or June and she was adamant, adamant I tell you, that we submit it within months. Deep down I knew this was never, ever, EVER going to happen. Why? Because the last two papers I wrote with this person took over 1.5 years to complete. Not 1.5 years until publication, 1.5 years until submission. Not surprisingly, I received her corrections in September. "Fuck this shit," I thought to myself. I put the paper aside, but for good reason.

At the time, I was knee-deep in expressing proteins and getting very close to finishing up a few major experiments for a paper in post-doc lab. I know how paper-writing goes in post-doc lab and suffice it to say, that I could finish data for this paper, write the paper, submit the paper and get the paper published (with time for revisions) before my paper with grad advisor was even remotely close to getting finished.

October rolls around and I get an impatient email from grad advisor, asking for my revisions. She claimed she had all this free time for writing and really wanted to get the paper submitted before the end of the year. She really thinks this can happen if I just send my revisions. So, I send her my revisions the following Monday and guess what, I haven't heard jack shit. It's DECEMBER! Um, that's pretty much the end of the year according to my calendar.

I don't really care anymore that writing with this woman occurs at a glacial pace, but it infuriates me to get rushed along with the promise of reward when nothing happens. Hurry up and wait, hurry up and wait. What's worse is that I fucking fall for it. Mark my words, this paper won't see the journal submission page before this time next year. In the mean time, I'll write, submit, revise and publish a paper from post-doc lab (barring any horrible issues arising).

Damn it! I'm all in a huff about this.

I snarfed

Despite the fact that I live in a state where you can't swing a bag without hitting a fundamentalist-type christian, I rarely discuss evolution with those that don't already believe it.
This Friday, I was enjoying time with friends when some new people (new to me anyway) joined our table. Inevitably, the "what do you do for a living" question came up and I gave one of my stock answers, "I'm a microbiologist." My friend then chimed in, "she has a PhD." Immediately the guy turned around and said, "Oh, well then I'd like your opinion on this awesome movie that I saw a couple of weeks ago." I figured I was about to get asked about a somewhat microbiology-themed movie, like 28 Days Later or Outbreak, so I said, "sure" and took a big ole swig of my beer while he formulated his question.
Guy: "I can't think of the name of the movie, but it was really informative and it had that Ben Stein guy in it."
Me: I snarf the beer. (snarfing = verb, when liquid that you put into your mouth comes out of your nose. You can also snarf a solid.) Finally, after I composed myself, I asked him if he was referring to expelled.
Guy: "yes!!" Followed by, "I just wanted to know what you thought about it."
Me "I think the movie is propaganda for the Discovery Institute."
Guy: "Oh, why?" So I tell him, and I am pretty nice about it. Eventually, he asks me why I don't think intelligent design should be taught in the science classroom, to which I replied, "There is no evidence for intelligent design and science is evidence-based." Then he said, "Well, there is no evidence for evolution either." I paused, shocked at the statement. I was prepared for comments about the gaps in fossil records and the like, not flat out denial that there is any evidence for evolution. Finally, I calmly replied, with a five or so pieces of evidence for evolution. Afterwards, he gave me the evil eye and started playing with his phone.
I think I need to take this as a wake-up call to get better prepared to talk to people. At the very least, I think I should avoid blowing beer out of my nose. I don't think it gave me too much cred.

It could be worse...

Typically, I can't stand it when someone dismisses my foul mood with the statement "It could be worse." But, no matter how much I dislike this weird version of a silver lining, I can't deny that it is true. (Of course, I suppose you can say "It could be worse" about any bad situation except terminal illness and death.) Just over a year and a half ago life handed me a fantastic example of how things could indeed, be worse.

The prelude to this lesson was the realization that my dissertation project was completely fucked. The person I inherited the project from messed up everything. EVERYTHING! I am NOT exaggerating, and just to prove it, I am going to provide a few examples of just how messed up it was.

1. We were working with an unsequenced organism and the locus we were studying was sequenced by my predecessor. The sequence was wrong. An examination of the records from our sequencing facility revealed that he only amplified and sequenced the locus one time.
2. He engineered his own plasmids, but placed the origin of replication into the multiple cloning site (MCS), resulting in bizarre and unreproducible expression of genes placed within MCS.
3. Strains labelled as single mutants were actually double mutants due to sloppy design of knock-out construct. (No wonder I couldn't complement them.)
4. Claimed expression levels of genes of interest were extremely low only to find out he didn't induce them.

Finally I realized that the only thing I could do to with the strains, plasmids, etc., was throw them in the trash, which I did. I went through every shade of mad possible and probably would have been arrested for assault if my predecessor hadn't moved to Singapore to wreak havoc in some other lab. I tried to move on and let it go, but I couldn't. This wasted over a year of my time and I was freaking pissed off. The fact that no one was here to pay for such sloppy work only added to my frustration. I started everything anew and things began to work, but that empty space I had on my shoulder was replaced with a little chip, that got bigger with any obstacle I ran into that I could trace back to the jack-ass before me.

A couple of months later I got an attitude adjustment by way of a friend who went from writing his dissertation to flying back to his home country without so much as a masters. This is what happened to him:
My friend, who I will call Carl, was working for a problematic PI. He had a hypothesis that he wanted Carl to prove, but all of Carl's data disproved the hypothesis. Carl's PI pushed him to repeat a certain experiments over and over, convinced that Carl was the problem, even though all the controls indicated otherwise. Eventually, Carl's PI tried to kick him out of school, citing that he was a poor student and was not dedicated to research. Fortunately, his committee disagreed and ultimately Carl was moved to a different project. (Yeah. Big red flag. You never set out to prove a hypothesis. You can only set out to test it.)
Carl's new project went smoothly and within a couple of years he produced enough data to write his dissertation. He also published a couple of first author papers and had another paper submitted (I think). Carl even interviewed for a great post-doc position at a somewhat prestigious lab and got it. Then all hell broke loose.
Before he could defend, his PI came under investigation by the NIH when he tried to publish a paper using a figure that was already published in another paper from his lab. This paper was not related to Carl's work and Carl was not on the paper. Everything from the lab was confiscated, including all computers and lab notebooks.
An examination of these things cleared Carl. All of his data was accounted for in his lab notebook and in logs from using equipment and core facilities at the institution. At this point, things seem like they are going to be OK for Carl. His data was deemed safe, he still had his kick-ass post-doc and he could continue writing his dissertation.
Unfortunately, the school decided not to let Carl defend his dissertation. The university would not even let him get a MS on the work that had been published previous to the debacle. Again, none of his work was related to the bad figure or paper. In the end, Carl spent six years in graduate school, got enough data to write a dissertation, but ended up with nothing to show for it.
As I watched all this unfold my project problems seemed seemed lame. At least I could salvage everything and none of the bad data from my predecessor ever made it into a manuscript. I was still going to get my PhD and I only wasted a year, not six. I flicked that chip off my shoulder and moved on. That doesn't mean I am not still pissed at the person who screwed up my project, but I don't dwell on it anymore, because it could obviously be a shit-load worse.