Seriously, who sends DNA…in non-sterile plastic tubing…that a plastic pipette tip cannot fit into…*epic facepalm*
Labwork has been frustrating lately. My favorite protein (MFP) has been less than cooperative over the years. First, it was a royal pain in the butt to purify to any reasonable degree. At best, I had to settle for a co-purified complex that was for the most part functional, but later to be demonstrated invalid. Last winter just after my last committee meeting, the original lab working on MFP published data stating that they have been working with a protein with an incorrect translation start site. In more
lamen’s laymen’s terms, it meant the protein I had originally purified was wrong; the protein was about 15 amino acids too long. So since then, I’ve been trying to get this protein re-established and purified using their new protocol so I can redo all my old biochemistry, and finally move forward with the new biochemistry I had proposed to do. Well, it’s been nearly a year, and now I think I finally have some hope of purifying it.
- Issues that I’ve had to address include:
- Poor expression (to the point I can barely see my protein above background cellular proteins)
- Inability of affinity tag to stick to affinity resin (or any resin for that matter, ion exchange included)
- Poor plasmid maintenance after an overnight culture (and have nothing left to grow the next day)
Since then, I’ve recently introduced a new selection marker that’s far less likely to be ignored that now seems to be giving me appreciable yields of expression in a rich, defined media (instead of our typical undefined media). So right now, I’m ready to crack open all my cells and see just how soluble my protein is. If it’s well-behaved in solution, then it’s on to the FPLC and see what I can get this sucker to stick to!
I also need to submit my construct for DNA sequencing; I really want to make sure that MFP’s coding sequence is pristine. No gaffes this time! I have to sequence a bunch of my selected mutants that have passed muster so far. Up to nearly twenty candidates and I need to get some sequence back to see what kind of mutations (if any) I am getting in MFP.
Additionally, since the “correct” translational start site is supposedly published now (nearly a decade since the lab originally identified the protein in the organismal genome), we’re not entirely convinced ourselves to run headlong into this and just trust it. I did get kind of screwed by trusting the original research in the first place, no? So, I had vocalized my concerns with this being an accurate piece of data, being as how they minimal upstream sequence to incorporate any endogenous promoter sequence. Well, that came back to bite me in the rear today; my advisor would like me to affinity tag my protein with an endogenous promoter sequence and get this guy purified and sequenced by Edman degradation to verify the start site. I don’t mind cloning, but I do sort of loathe designing primers for PCR. So that’s my new side-project to verify everything is kosher, so to speak.
The genetic selection library is coming along nicely, but the undergraduate who is working on the screening is a different story. I’ll save that for another night, so I don’t unnecessarily rip on her. She does a good job, don’t get me wrong! She just tends to miss some crucial steps on the way…or a lot…yeeeeeeeah.
Just recently, I decided to attempt to pick up some manuals and tutorials in order to instruct myself in the ways of Cocoa application construction. Unfortunately, going into this I had no idea how to understand the underlying programming Objective-C in Cocoa. After picking up a text from the local B&N, I’ve found that going through it that it is not all that different syntax-wise from Perl and my really old days of C++ (shoot, about 7-8 years ago I played with teaching myself?). The coding syntax is great…I just need to get my head wrapped around this object-oriented programming so I can visualize this while I code. It’s a bit foreign to me this policy of using classes and objects for modifying data and/or sets of data, but at the same time it all sounds pretty cool.
This endeavor to teach myself how to program for Cocoa stemmed from a problem in the lab that I’m trying to negotiate and remediate. We have a massive collection of bacterial strains frozen down in our -80˚C storage freezers. In order to keep track of everything in there, we obviously have some kind of log/database for this. Over the years, the electronic database that was established has become more and more unkempt due to the flawed design when they set it up. The file was originally setup as a flat-file database in FileMaker, and the searching capabilities of the database/software is horrendous. It’s nigh impossible to find anything in the database unless you enter just the exact piece of text. So, logically, we want to clean this electronic database up to a standard we find fit (everything is recorded by hand as it’s created in a massive binder as a working hardcopy) with adequate amounts of information and good to exceptional searching capabilities (basically searching any string/entry for a piece of text).
Playing around with some other software, and reading up on relational databases, it seems that FileMaker was just an overkill piece of software for what the lab was using it for…a flat-file spreadsheet. It’s a relational database tool, and it was far from being used as such. Granted, I could try to reconstruct a FileMaker database and beat my head against a wall until I found out how to properly integrate a search function into the database, but that just would not be any fun! FileMaker/Apple’s home-entry database software Bento is a really simplistic database tool, using a simple flat-file setup with a bunch of flashy Mac OS X 10.5 features, as it’s Leopard-specific software. The searching in this software is fantastic for how we query it to find our entries, but it lacks so blasted much in what we want the database to eventually do. I’m thinking this will serve as an interim tool for managing our database until option three has made itself ready.
Yes, option three is me trying to write a small Cocoa app that will create, manage and interface with the database. I’m relatively impressed with the Core Data capabilities to date, just in what I’ve seen from Bento and iBank (despite iBank’s sluggish response when its database gets large) and I’d like to utilize it if I could. In addition to all this, I just think it’d be far more fun to write my own application for this kind of thing. I can ultimately add or remove features at will (provided I don’t spontaneously forget how to code it), and it’s free! Well, it’s costing my time, but it’s time I’m wiling to give up for it. I think I’m comfortable enough with the Apple platform that I’d be willing to learn how to write small apps for it to carry on with me over time. It’s just a matter of taking the time to suck it all in, get my head around object-oriented programming, and then learn how to interface it all with a user interface. That last part is what I think may ultimately kill me…I sure hope Xcode has some wimpy simple tool for building and interfacing with a UI setup.
Luck will have it, I’ll get this all finished before I graduate, and then Apple will restructure Cocoa so much that I will not have a clue what I’m doing anymore in it. I’m crossing my fingers that this doesn’t happen!
P.S. I don’t think there’s enough researchers out there willing to write their own applications for tools…at least I never meet enough that aren’t already computationally-focused.
I’ve been a bit busy lately dealing with a lot of real life stuff. This week has kind of been a symposia week, even though there’s only two that I’m attending. Just nailed one out of the way today, with one more to deal with on Friday. I’ll be presenting my own research then, so I really wanted to make sure that I had a decent presentation for it. I really don’t feel it’s as comfortable as I would like, but I don’t think I have much of a choice considering how my work has progressed lately. The ChIP isn’t moving along as nicely as I would like, but that’s another story for after I’m done stressing over symposia and talks and such.
For those that actually peruse this blog, look for a post in a day or two once I’m free from extracurricular obligations.
The weather has gone crazy again. Big surprise, right?? We had beautiful, sunny skies yesterday with highs somewhere in the 50’s, and now we’re down to crazy mid-20’s with freezing rain and snow due later tonight. Crazy weather fronts and silly southeastern moisture!
This weekend is recruitment weekend for my graduate program (Interdisciplinary Graduate Program in the Biomedical Sciences). Like the weekends in past since I was accepted into the program here, I’m helping out with the recruitment process by meeting with the students and answering/reassuring any questions they may have. Personally, it’s nice to meet some interested parties in the university and help get them excited about coming here, but I have to admit that the free food and beer is a nice perk on the side also! Thursday night is casual night out to break them in and let them meet some students. Friday they get to see the school and meet with selected faculty members of their choice, lunch with students and faculty both, and dinner with faculty and students again. I get to help out with both dinners, and then meet with them one last time Saturday morning for breakfast to show those interested about general housing areas in the city and ‘burbs. Granted, I’m no expert like many of the people who grew up in this region of WNY, but I’ve gotten a good feeling over the years for what’s nice and what’s sketchy.
Being that this is my third year helping out with the recruitment process, I’m starting to feel my age within the program a little bit. The first year I helped out, there were a couple fifth year students still doing it, and numerous other ones above me. Since then, we’ve lost more of those upperclassmen. This year, there are only three that are upperclassmen to myself, and only by a single year. I’m getting to that point that I am the upperclassmen, and it’s starting to feel weird. This is only compounded by the fact that I’m no longer taking real coursework (just research credits and seminars), and I’m already on my way to candidacy as long as my research continues to be fruitful. Ironically, I don’t feel that I’ve accomplished a ton in terms of research, but I have gotten some really interesting results since I’ve started my project. Additionally, I’m on the edge of finishing some crucial experiments to determine where my research is going to continue, so perhaps I’ve really accomplished more than I thought? Who knows…I’ll just be excited to finally get a project of my own to publish!
It’s late on a Saturday night, and I just don’t feel like going to bed quite yet. Despite
nearly being done with my BerryWeiss and developing a mild headache (likely induced from unnecessary screaming at a lacrosse game), jut not in a hurry to get to sleep yet. I’m sure that will change once the temperature has dropped and I start getting cold.
The Bandits game tonight was good once they got their act together. I’m so used to seeing Kilgour as the starting center, that I didn’t even think twice when the away team (Philadelphia Wings) got the ball repeatedly after scores were made. Only then did I notice we had some rookie Sanderson at center, and he was doing a horrid job in the face-offs. Swung wild nearly 6 times, completely missing the ball and giving it to the Wings. Kilgour would fight until him and the opposing team member would go down on the turf before he’d give up his hold on the other man’s stick. Within 7 minutes, the Wings were up 6 on our nothing going into the game. I couldn’t believe it! Then the one chance we did have to score (and theoretically did), they called a roughing penalty on the shooter and negated the score. I was a bit livid, since it appeared to be a shoddy act more than a real blow to the goalie.
Ironically, once we got most of the rookies off the field and went back to the tried and proven guys, we came back…with a sweeping lead I might add. Game ended 21-12, thanks to some cooperation resultant in some real shooting (1st quarter shots were lethargic and horribly inaccurate) and a solid defense. I was very glad to see them come back and clean their act up.
Work was a little stressful this week. The week was going okay up until Thursday morning. Wednesday night, I left our FPLC unit running with a chromatography column on it that I was equilibrating for the morning. Unfortunately, I forgot to turn down the flow rate and it depleted all the water I was passing over it, resulting in approximately “420mL of air” going over the column. Needless to say, the column dried out, and the pumps were running dry with dried out seals, no less. My boss was the first one in Thursday morning, so he was the one to discover this first, much to my chagrin. Thankfully, being the ridiculously laid back fellow that he is, he wasn’t too flustered by this. Just caught off guard by the odd noises coming from the machine. The column has since been rehydrated, and is still flowing under a constant flow of ethanol/water to ensure there is nothing problematic with the column. It will continue to run like that all weekend, and then I’ll run some standards on it on Monday (providing our rotation student isn’t using the FPLC) to see how badly I’ve affected/destroyed the theoretical plates in the column. I’m praying it’s minimal…the column runs around $1400 brand new! Then again, I’m not sure I’d want to purchase a used one. Regardless, the FPLC is running fine, and hopefully the column will be as well.
The cat is starting to pester me, so I think it’s time to head to bed. She gets a little too whiney in the middle of the night for attention, I swear!