It just might work this time \o/

The situation: I'm slated to present a poster (blech, I hate posters) at an upcoming meeting. When the meeting is two weeks away, the clouds part, the sun shines and all my cloning problems are resolved.

The question: Now that the constructs are complete, can I get the strains made in enough time to do experiments and include the results on the poster? (I've wanted these results on the poster all along, but after I stumbled into a bad cloning patch, I resigned myself to leaving this data off.)

The Answer: If I work everyday between now and the meeting, don't make any mistakes and find a way to print my poster at the 11th hour, I can include the data.

Sidebar: I consider throwing in the towel before I even attempt this whirlwind of tasks. You see, this shit happens all the time. I have a breakthrough within two weeks of an important meeting or presentation and bust my ass trying to acquire new data to include on a poster or in the presentation. All signs point to things not working out, but I try anyway and attempt to just push through only to finally realize a couple of days from the deadline that there is no possible way to get everything done. As a result, I am exhausted, irritated and consider my poster or presentation completely lacking as it doesn't contain the new data.

Current Standing: Well, I didn't throw in the towel. I am t-5 days from the conference. The strains are made and almost all the experiments are complete. By the end of today I should actually be in a place where I can put the poster together. If things work out, this will be the first time that last minute data gathering doesn't end in a complete and total cluster fuck.

My big news in the form of a riddle...sort of.

If a game of ping pong represented funding, and I was the dude in blue, what would you guess happened to me?

If you guessed obtain my own funding, then you are correct. I actually received the good news back in June, but I didn't want to write about it until I actually received the fellowship money.

And yes, I did dance around just like that guy.

Solutions for solutions

Apparently my current lab contains people who cannot or will not order supplies. As a result, we are constantly running out of certain regularly-used items. I know that this problem is not unique to my lab. On a good day, finding a certain reagent or supply completely gone makes me mildly annoyed. On a bad day, this can send me into a rage.

Yesterday, I found myself annoyed as I was having a decent day already. A couple other co-workers and I discussed the ongoing problem and potential solutions. My solution, regrettably, was to start hoarding this particular supply (one that I always need and we are regularly out of) for myself. I really don't like to hoard supplies, but I also don't like for my experiments to get put on hold because some dumb fucking donkey couldn't fill out an order form.

This ultimately lead to a discussion about people taking your solutions and reagents without asking and the remedies we each had employed in the past. (Note: I am NOT talking about a few mls.) My co-workers had tried (1) hiding solutions; (2) writing "DON'T USE WITHOUT ASKING" on the solution label and (3) giving solutions generic names such as "A." My remedies included making the solutions weird concentrations that require the thief to do math. (For example, 1.41M instead of 1M.) The hypothesis here being that if you are too lazy to make solutions, then you are probably too lazy to do math. If this doesn't work, I mislabel my solutions. The label may read 1M Tris, but it is actually 5M NaCl. This is most effective because the thief determines you can't make solutions and quits "borrowing" from you. I was told this was kind of evil, but I think taking people's shit without permission is still worse. Of course, I am an only child and I don't like to share. :)

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.

Today was not the greatest day I've ever known

Right off the bat, I discover that my dialysis tubing leaked and my beautifully purified protein occupied a 2L volume. I first tried concentrating it, which worked at first until the apparatus broke. After that, I just loaded the remaining 1100 ml over a column.

The unfortunate part is that this protein is one in a set of five (a parent protein and four mutants) that I purify simultaneously. I prefer this approach because it ensures that the proteins are subjected to almost the same conditions. These proteins are very sensitive to purification and storage conditions, including length of time spent in storage, so it's really not ideal, in my opinion, to purify this protein by itself. So, if this protein isn't salvageable, I'll probably start over again with all five. Ugh. It's not the end of the world, but I am irritated because this dialysis was the final step and means I essentially wasted the majority of this week.

oh. well. breath in, breath out, move the fuck on.

I'm still here...

I've just been way too damn busy to write any blog posts and even worse, I haven't gotten to read any blogs either.

Anyway, I decided last week that this was going to be the week where I got my ass back into gear and started blogging again. Of course, the week before last, I had decided that last week was going to be the week, but clearly, that didn't happen. As I neared midweek, I was starting to think that maybe I would push things back again, but thankfully, I find myself at work, in between experiments with some time to blog.

I can't believe how insane things are right now. I've been purifying proteins like it's going out of style. Only four more to go at this point. I can't wait until an entire two-week period passes where I don't even so much as look at the FPLC. I'm sick to death of it.

On the plus side, I made some headway with the boring paper from grad school lab and I am about to start writing the paper that caused this protein purification extravaganza. I'm pretty excited to get a paper out under less than a year, but I am freaking exhausted.

During all this time away, I started writing my specific aims for the F32, but I didn't get very far. I really need to get moving on the grant writing, but it just keeps getting thrown to the back burner. Maybe next week...

Hope everyone in the blogosphere is doing well. I can't wait to catch up on everyone's posts.

Is there really no such thing as a stupid question?

I am not sure I believe that there are no stupid questions. During the last couple of weeks I’ve fielded quite a few that qualify as such.

The first example comes from the people working on renovating my condo. Unfortunately, most of the questions they ask qualify as stupid for the simple reason that I have already answered them multiple times and in multiple ways. This most recent question that I had already answered involves paint. Per their request, I picked out the paint for the ceiling, wall and trim in the hallway, paid for it and brought it to my house. I placed the cans of paint in the living room, inches away from the path that the men will take to get to the hall, and labeled the tops of the cans “ceiling,” “wall,” and “trim.” Yesterday, I arrived home to find a laundry list of questions including, what color do you want us to paint the hall and where is the paint?

The next example comes from the people who are conducting some animal experiments that I need for graduate school paper #2. I sent my strains to our collaborator with documentation describing the genotype and phenotype of the strains, including a formally written note included with the shipment, an email informing them that I sent the strains and what they should expect to find in the package in addition to labeling the actual strains. This is what you need to know about the strains:
Strain 1: ampicillin-resistant
Strain 2: ampicillin-susceptible, kanamycin-resistant

A couple of days after our collaborators receive the strains, I get this message:
Microbiologist XX,
I think something is wrong with the strains that you gave me. I streaked them out on ampicillin, but only one of them grew.
Sincerely,
Animal Collaborator

I respond with a nice email explaining that every thing was fine and only one strain should grow on the ampicillin. I also describe the relevant characteristics of each strain again.

A couple of weeks later I get this email:

Microbiologist XX,
Are you sure that both of your strains grow aren’t resistant to kanamycin? When I streak the amp-resistant strain on plates containing amp and plates containing kan, it only grows on the kan plates. When I inoculate the strain into broth containing kan or broth containing amp, it grows in both. Are you sure that your amp-resistant strain isn’t also kanamycin resistant?

Again, I respond with a nice email explaining that no, there is no way that ampicillin-resistant strain is also resistant to kanamycin. I tell them that I think they are introducing a contaminant. I ask them to check the original stocks for growth in the presence of amp or kan and to let me know if the strains grow in the presence of an antibiotic they shouldn’t grow in. I haven’t heard anything since.

I don’t mind answering these questions. In fact, I am glad that they are asking me questions when they run into problems, but at the same time it worries me. If you can’t keep a stock or culture free of contaminants then how can I be sure that the data you send me reflects my strains and not my strains plus some other random contaminant? Is sterile technique really that hard?

The age old question...

Over the years, many people have asked me the dreaded question, "What do you do for a living?" Throughout graduate school the answer continued to change on a somewhat regular basis. The first thing I learned was to avoid informing others of my student status. I found it difficult to explain to people that while yes, I was a student, I really didn't spend that much time in class and that instead, I spent most of my hours toiling away in the lab. The additional benefit of not telling people that you are a student is avoiding the painful, "When are you going to graduate?" question. People just don't take well to "I don't know" or "It depends."
Even after deciding to remove the word student from my answer, the response to that question still changed over time. Versions included:

1. "I am a microbiologist and I study gene regulation in Painintheass bacterium, the causative agent of horrible way to die disease. Followed by way, way too much information."
2. "I am a microbiologist and I study gene regulation in P. bacterium. It causes disease X."
3. "I am a microbiologist and I study P. bacterium. It causes disease X."
4. "I am a microbiologist."
5. "I grow and kill a shit-ton of bacteria on a daily basis." (current response)
   

It's not that I don't like to talk about what I do. I freaking love, love, love to talk about my work, but molecular biology is just a little to complex for the lay person and I am getting progressively worse at using non-science lingo. I find that people want the short answer, and for me (at least for now) that is #5. If people inquire further, then I provide more information. For me, the take-home message I learned about discussing my career with non-science geeks, is to let the person you are talking to determine the amount of information they receive.

Does anyone else have their own version of #5?