I’m all kinds of wiped out this week. Some long (albeit productive) days combined with poor sleep and a return to strength training have sapped much of my will to live.
Been troubleshooting the fluorescence anisotropy by trying to verify that my DNA oligomer template is even being bound by my protein of interest. No biggie; let’s just do an EMSA. Well, that turned into a small mess of issues:
- The fluorescent oligomer (40-bp) is way smaller than the normal control DNA fragment (341-bp), and so runs off the gel under the typical 5% acrylamide native PAGE.
- The fluorescent oligomer didn’t even look to be properly annealed.
This week has been trying to resolve what the discrepancies are for annealing. Conveniently, the oligo is fluorescently labelled; I need no stains or dyes to visualize with. Inconveniently, I need to run out native PAGE to assess the bound/unbound state of the oligomer pair. Anyways, it basically boiled1 down to bad primer concentrations2 and optimization of annealing conditions3. Troubleshooting resolved; I can move on to the real experiment now!
Sleep has been slightly lacking this week. Although it hasn’t hit me too hard, I am way behind on it. Tuesday night I didn’t get to sleep until almost 1, and my body decided to wake me up against at 4ish, wide awake. I thought Wednesday was going to suck. Thank god I powered through the brunt of the day before charging up with some free coffee immediately prior to & after the lunch seminar. I had hoped to get to bed early last night to start to catch up, but cooking ate up much of the evening. I wonder if there is anywhere kosher I could curl up in the sun at this weekend to nap? I don’t have this luxury in my present apartment anymore, so my ability to nap is nigh ruined! If I look for a new apartment this coming spring, I’m definitely trying to find one above the garden level, or at least have some proper windows if nothing else!
Strength training, egad. It’s not much as of yet (squats, pushups, straight leg raises), but trying to work through three sets has been awfully rough. The squats aren’t too bad4, but the pushups are worse than I was expecting. I was doing mid twenty-ish of them back in the spring (when I also weighed more like 240 than 180) in a single set, but right now I’m struggling to finish the ninth and tenth of my third & last set; like almost face-planting myself into ceramic tile struggling. The raises, dear god, I don’t know if it’s my legs or my abs that can’t handle it; I’m guessing my lower abs (from the aches), but my upper quads are also barely holding out throughout the lift. So bad in fact, I can’t do them as frequently as the other two just yet. I’m rotating the exercises in three-day sets: first day is 3×15 squats, 3×10 pushups, and 10/8/6 leg raises (hoping to get them to 3×10 soon); second day is just the squats & pushups; third day is the rest day
Although, for my rest day today, I took a three & a half mile mid-day walk for a break5, and an unexpected five mile walk this evening. I intended to walk to Dark Horse Espresso on Spadina to finish working on data/lab notes from today, but they were plumb full up on seating. So I kept going and took a trip to Valhalla Cards to pick up some small notebooks for jotting experiment plans & such on while I’m riding to/from work on transit now. Since the Queen St streetcar was still looking like packed sardines, I just opted to keep walking to Lansdowne. Except I walked the wrong direction. I paid no attention to my orientation when I left the store, and back-tracked all the way to Spadina Rd—an entire kilometer at this point—before I realized I was heading the wrong direction. Turning around & walking back towards Lansdowne, I had put on a full five miles when I got to the start of the 47 bus line. I was exhausted.
I still am, for that matter. So on that note…
1No pun intended. You know, since you have to bring the DNA primers essentially up to boiling & slowly cool them to promote proper base-pairing of the primers.
2My bad for not checking those early on; I’m too used to just using primers for PCR & not for more refined purposes.
3Inverted repeat sequences are apparently good at forming hairpins. Duh!
4Considering I was doing them most of the summer after my rides home, the leg part of the squats isn’t that bad; my arms & core are more of where I feel the fatigue.
5It was a beautiful day for a walk today, wow. The wind was hardly what the forecast anticipated & the sun was shining!