by PhilipJ on 14 December 2005
Having been inspired by Chad’s excellent week of blogging from his experimental physics lab, and some complaints by a commenter on Cosmic Variance that us (non-particle physics) experimentalists don’t blog about our daily routines nearly enough, I’ll tell you all about my day at the lab yesterday.
Okay, okay, I jest, it wasn’t that I sat around twiddling my thumbs, I simply had one of those days where the laws of physics seemed to break down in a very specific area called The Lab I Work In. Here’s how.
I have built a single-beam optical tweezers apparatus, and have been working in our collaborators wet lab to get the molecular biology side of my project going, but back in the physics lab we had yet to do a crucial first step in terms of biophysics experiments, that is to pull on (and measure the force extension relation of) double-stranded DNA (dsDNA). Here’s a shot of my tweezers in action, with 3.17 μm diameter beads held in the optical trap (left) and on a micropipette (right).
The obvious way to do this is to simply stick a piece of dsDNA between the two beads, and move the pipette relative to the optical trap to apply forces, all the while measuring the bead-to-bead distance. This is perhaps easier said than done, but the basic recipe to follow is: make DNA with special linker molecules on either end (in our case we’re using biotin and digoxigenin) and coat the beads with complimentary binders (streptavidin and anti-dig, respectively). Put the DNA and one set of the beads together to let them bind for a while, and then try and “fish” for a piece of DNA using the other type of bead stuck on the pipette. If chemistry still works as it is supposed to, the DNA’s free linker will find a binding site on the other bead, and get stuck between the two.
Sounds okay so far, but there is one crucial component of the system that is a little finicky—the micropipettes. The very tip of these pipettes are quite small (roughly a micron in diameter), and you can imagine they can fairly easily get clogged if care isn’t taken. You also can’t, as far as I know, buy them commercially (hi patch-clampers, your pipettes are much too big!), so we pull our own here in the lab. Eighty micron outer-diameter glass capilaries get fed through a small loop of platinum wire, and are clamped so that the top is immobile, while the bottom is pulled on via gravity. As we ramp up the current through the wire, it starts getting quite hot, and eventually the glass melts and gets pulled away from the loop via the weight on the bottom. By tuning the speed at which the loop heats up, you can pull pipettes with varying tip sizes, and we spent quite a while figuring out the right parameters necessary to reproducibly make pipettes of the size we need.
Here’s where the laws of physics seem to have changed on me yesterday. Even though our current ramp rate was the same as always, the same loop of platinum wire as always, the same glass capillaries as always, none of the pipettes I pulled yesterday were the right size. I’ve been doing this for months with fairly reproducible results, and yesterday, on the day I was going to finally get a force-extension relation for dsDNA (about which we’ve had a half-dozen other non-trivial problems getting to work), the pipette tips were all garbage. Some were too wide at the tip, others had asymmetric tips that would, by residual suction, gather a whole cloud of beads around the pipette. None were suitable for doing single-molecule biophysics.
Luckily, the laws of physics seem to have returned to normal at my lab, and I’m going to try and get that elusive First Data (even if it is just for calibration purposes) today. If all goes well, I’ll post a bit more about what dsDNA force-extension relations are all about with some real data soon. Wish me luck!