Yep, it's been that kind of day.

I started the day off well enough. I woke up on time, I made myself some coffee (bonus) so that I wouldn't need to stop on the way. I get in the car and proceed to work, quite proud of the $1.50 I am saving. However, about 30 seconds later, the universe decided that today, I would provide it with comic relief or something.

Here is a break down of the events - and I'm just hitting the easy-to blog about items. Just imagine how my experiments went today.

1. Sweet ass (not the good kind).

While drinking my coffee on the way to work I realize that my coffee tastes awful. WTF? I ask myself. After another sip, I come to the conclusion that the giant container of half and half I just purchased isn't plain, it's french vanilla. GROSS. My coffee tastes like sweet ass!

2. My boob is really wet and warm.

I decide that my need for caffeine far exceeds the taste of the coffee so I continue to drink it. I'm having trouble drinking from the travel mug and decide there must be something wrong with the spout (or whatever that slide-open thingie is called). Oh well, slow coffee is better than no coffee. Right? Several labor-intensive sips later I notice that my left boob is feeling quite wet and warm. OH. MY. FUCKING. GOD. The coffee is leaking from under the lid and is now all down the front of my shirt and has apparently, soaked through my bra.

I consider going home to change, but cultures are waiting for me and my shirt is navy, so I ignore the signs of the shitty day to come and proceed to the lab.

3. Somewhere between 2X and 3X.

I arrive at my bench to find all the media that I prepared yesterday. The best thing I can say about the media is that it is sterile. Unfortunately, it also looks like it sat in the damn autoclave all night as the final volume is less than half of the starting volume.

I'm starting to feel a tantrum coming on, but I can find no one to take my rage irrationally out on at this point (lucky bastards) so I take a few trillion deep breaths and continue on with my day.

4. The sink stops working.

5. Fucking crystal violet!

I'm nearing the end of an adherence assay and start making the crystal violet solution. This go well until I try to filter the solution. That's when this happened:

Then I remembered that I was wearing relatively new shoes.

Five minutes into cleaning the mess.

Drink please.

Dumb F**king Donkeys Strike Back

I’m sure this is common in many labs, departments and offices: Some dumb fucking donkey (DFD) takes advantage of a good situation and everyone suffers because the people in charge prefer punishing everyone instead of confronting the person who is actually causing the problem. These instances might include, but are not limited to the following examples.

DFD situation #1:

DFD takes advantage of ordering by claiming that PI approved the purchase of an expensive reagent or kit (or whatever). After the item is ordered, it is discovered that permission was never granted and PI was completely out of the loop. This happens a few more times and involves the same person.

Solution: Enforce a new rule where ALL orders must gain approval from the PI, causing orders to get placed only once per week as PI does not have time to review orders multiple times a week.

DFD situation #2:

DFD refuses to turn in packing slips when an item arrives. This of course, pisses off the purchasing department, ultimately resulting in the lab manager getting bitched out.

Solution: Instead of speaking to DFD, the entire lab gets a 20 minute lecture on packing slips, what they look like, what they are for, why they are turned in to the purchasing department and what happens when they don’t. The tone of this lecture is extremely condescending and most lab members leave irritated because they don’t appreciate being spoken to like a child. As expected, DFD seems clueless and the problem continues.

DFD situation #3:

Instead of working a full day, the DFD comes in late and leaves early. Unfortunately, this DFD is the lab manager, so ordering is delayed; work orders for equipment repairs are put off, sterile items and media run out, etc. Lab members complain to PI.

Solution: Instead of asking the DFD to work more than 4.5 hours/day, the lab chores that DFD doesn’t want to do are redistributed to the lab members that actually work, i.e. post-docs and grad students.

Personally, I don’t understand why it is so difficult to confront the person who is causing the problem. I’m learning to take this shit in stride and file the experiences under, “Behavior I will not repeat when I obtain my own lab.”

Any DFDs in your lab/office/whatever

More time please?!?

Magnum, PI is very supportive and has no problems with his post-docs starting families. In fact, he was quite excited when I told him I was expecting and immediately told me not to worry about any decreased productivity that I might experience during the time before and after I gave birth. He went on to tell me about several other female post-docs that started families while in his lab that were able to balance work and life and move on to successful careers. This was wonderful news and it was a relief that he was fine with and understood that a decrease in productivity would to occur. Unfortunately, I was not fine with this then and I am still not fine with it now. It nags at me.

The fact that my project went to hell in a hand-basket this summer does not ease the constant nagging voice that tells me I am falling behind, that I am not producing enough data, that I am not getting as much done each day as I possibly can. I know that I just can’t work the hours that I used to, yet I refuse to except that this means that my productivity must also slow down. (I know, it's silly, but it's how I feel.) As a result, I’ve tried to make up for the lack of time in the lab by trying to utilize every single minute of time. I make plans, I organize accordingly and I multi-task like it’s going out of style. Unfortunately, I still find ways of not getting everything done and it drives me crazy and makes for a cranky MXX.

Take today for instance. I chose to stay home for a few hours this morning so that I could write and submit an abstract for ASM. I can’t seem to get a lot of writing done at work due to constant interruptions, but by staying at home, experiments are not getting done. I prefer to write the abstract at work during incubations, PCRs, etc. I tried this initially, but it didn't work out. On to plan B; work on the abstract at home during the evening. Unfortunately, my husband is working around the clock this week as a major deadline is approaching for him, so all child care falls 100% on my shoulders. Mini-xx goes to sleep at 7:30, but that’s when the never-ending chores start. After that I just want to go to pass out. So, on to plan C; take a few hours at home and push the abstract out the door while simultaneously NOT doing experiments. This is what I did, but I feel like it wasn't the best use of my time. I always think I can be doing something else while writing, especially something as lame as an abstract.

Anyway. The decreased productivity is constantly on my mind. I recently brought this up to Magnum, PI during a brief conversation. I stated to him that I was not pleased with how far behind I had fallen regarding my project. Specifically, I anticipated a paper being submitted at this point. He looked at me like he thought I was crazy and told me he was more than pleased with my work since joining his lab. He told me he thought I was too ambitious and that my expectations were slightly unrealistic. I’m glad that he is happy and it’s great that he is pleased with my progress, but somehow this doesn’t quiet the voice in my head and unfortunately, I still can’t see how my expectations are unreasonable.

To me the expectations aren’t the problem, I am. Cutting myself slack is not something I have ever been able to do. Instead, I’ve decided that I need to find a way to add another 5 hours to my time in the lab…at least until I get back on track. Putting in an extra hour or two everyday is not going to work. This would completely fuck up things at home and besides, I need a chunk of time. So, I’m thinking that if I can stay until 10pm two days a week (instead of just one), then maybe I can begin to get back on track. The downside is obvious, I will miss another night with mini-xx. On the plus side, it will keep me from putting in serious hours on the weekend, which I don’t want to do as these are the only two full days I get to spend with my family.

Oh I don't know. Now that I am reading what I've written, it does sound a bit crazy.

Good riddance 2010

Although 2010 brought great enjoyment to my life; the birth of my first child in April and submitting a successful post-doctoral fellowship application, Thanksgiving to Christmas sucked some serious ass. First of all mini-xx got sick and then my husband and I got mildly sick. Not that big of a deal. Just sore throats and snot. He chose to stay home for a few days, while I stayed home for one day. By the end of the week his mild sickness was gone and I was sicker than ever. I ended up staying home another three days with a horrible cough that didn't fully clear up until around Christmas. During that cold medicine-induced haze, I made and totally fucked up a reagent that is essential for an assay I am performing. Of course, I didn't realize at the time I messed it up. Oh no, that realization didn't come until the end of December after multiple assay failures. On the one hand - it was an easy fix. On the other hand - it was a stupid mistake and I feel like an idiot.

So far 2011 is going well. I am NOT coughing my ass off and my assay is working again! As a result, I should actually generate data again by the end of the week.

Although meager, the statement above is enough to make me pretty happy, but Magnum, PI recently informed me of something fanFUCKINGtastic. He is going to get me an anaerobic chamber. This is huge, because I need this piece of equipment to get my project for my own lab moving. No more band-aid solutions!!

I hope that everyone else is going strong on day 10 of 2011.

The lambs are still screaming

Holy. Motherfucking. Crap.

I don't know if I mentioned this before; I am claustrophobic. As a result, I don't particularly care for small spaces like elevators, closets, etc. I tolerate them because I know that I can leave. However, if you want me to freak right the fuck out, just make it so that I cannot get out.

Today, I got stuck on the 37 degree room. I went in to put some cultures on a shaker and when I tried to exit, the door would not open. Immediately, I started to panic. I wildly yanked on the handle and pushed on that lever that is supposed to release the door. It didn't work. Suddenly, it smelled like a million liters of E. coli were growing and the temperature in the room spiked by about 10 degrees. I could feel my sanity slip, slip, slipping away.

Just before I started screaming and beating on the little window like an actual mental patient, I gave calm and collected one more try. I turned the handle and what do you know...the door opened. I got the hell out of there.

The incident is over, but I feel on edge.

I need a drink.

Some times you feel like you have the force. Other times, not so much.

When I first started working at post-doc institution, I felt pretty awesome. Experiments were working well, Magnum, P.I. was impressed with my performance, etc. Things were moving along quite nicely. During this period of bliss, I somehow became synced up with the building elevators. Almost every day the elevators doors would part just as I approached them. It was one hand wave away from using the force.

One year and 8 months into my post-doc, things are a bit different. I'm in the middle of a cloning debacle and it is seriously starting to wear me down. The building must sense my ineptitude as the elevators have ceased cooperating. It's not just that the doors don't open when I get to the elevator. The doors are already open when I get to the elevator, but when I choose to step through them, they decide to close and ram me back and forth like a pinball. It's like the building is mocking me. Not to mention, it kind of hurts.

Of course, I won't allow my ass to be kicked by an elevator. The simple solution is to just press the up button before walking onto the elevator, even if one of the elevators is already available (taunting you). However, it was way more fun when I could pretend I was using the force.

Argh.

Sometimes I think the universe is trying to determine just how far I can be pushed before I completely lose my mind. I can tell you right now, it’s not that far. In general, my fuse is pretty damn short. Patience is not a virtue I possess and I do not fear or avoid confrontation. However, I really do try to keep myself under control, especially at work. If you compare myself of today with myself of 10 years ago, you would find significant improvements in this arena, but if you compared me to myself of just one year ago, you would probably find little improvement.

Then there are weeks like this, where you might find it difficult to see any progress no matter which version of myself you compared me with. It feels like 10 million little things are going wrong. No, none of them are worth losing my mind over, but as they add up, I find myself dangerously close to the breaking point.

As an example, let me describe a 1-hour portion of my day yesterday:

• Our lab possesses multiple thermocyclers and right now, we are down to one. I think fixing one of them would be nice, you know, just in case that last one decides to die. Yesterday, the last thermocycler died. Fortunately, it was some sort of issue with a fuse and was repaired quickly, but considering I cannot do what I need to do without PCR, I was pretty damn irritated.
• I send DNA for sequencing. I get results using one primer, but no results using the other primer. I use the sequencing facility's primers. If it were my DNA, then neither primer should have yielded a result. Therefore, I think it is their primer and that they should re-run the samples for free. They are not under that impression.
• I open the -80 C and a mountain of tubes fall all over the floor. Someone decided that balancing an open box precariously near the front of the freezer was a dandy idea. I want to leave their stupid shit all over the floor, but I pick them up and shove them back into the freezer.
• After picking up all the tubes and replacing them in the freezer, I try an locate the strain I need, only to find that whoever got into the stocks previously completely rearranged all the boxes and since they are covered in frost, I have to remove them one by one, open them and pull out a vial to determine which box I am in. No, I don’t fix the order, because I am annoyed as shit.

This kind of irritating crap has happened every day this week. I am so ready for the weekend. I need to rest and reboot. I really hope next week is significantly better.

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.

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.