User:Steven J. Koch/Notebook/Kochlab/2009/07/13/half micron beads; possible tethering
Half micron beads
Steve Koch 22:54, 13 July 2009 (EDT): Really great news from 0.5 micron beads. They trap really easily, are easy to see. We got really good looking power spectra from the beads, and it was good news. At 39.95 mW, had about 0.0047 pN / nm, so about 0.12 pN / nm / W. This is still lower than with old OT, but it's probably good enough for unzipping.
Steve Koch 02:47, 14 July 2009 (EDT): Quick notes. We were able to find many jiggling beads. This was using Ant's pBS___ #1? sample. I wouldn't say they had uniform starting lengths. Furthermore, there weren't a lot of them or many stuck beads at all, so it could have been non-specific. I would say there were about 1 tether every 10 eyepiece fields of view. Protocol:
- antidig thawed and diluted today with very old PBS + tween. the tween could be a very bad idea! Need to make PBS
- DNA from Ant diluted 1:100 in Brigette's BGB Popping buffer
- BGB from Brigette
- Beads incubated for 7-10 minutes. Beads diluted from stock 1:110 into BGB Pop, and sonicated in Evans' batch sonicator--beads were quite nicely monodisperse. Did not wash the beads, thus free streptavidin may be an issue
Data files are in "Shotgun DNA Mapping" directory.
Steve Koch 03:07, 14 July 2009 (EDT): We were able to get more power out of the power controller by putting the ND filter all the way to the 3rd "in" position and resetting the device. I can't remember the exact number, but I think we may have gotten up to 1.7 watts, but then the device maxxed out again. So, it looks like maybe that's as high as we can get unless we take the power controller out.
Steve Koch 03:07, 14 July 2009 (EDT): The closest thing I saw to possible unzipping was when Linh and I seemed to see the bead get stretched far and then recoil. However, it was not repeatable and probably not worth remembering. The data showed some strange hysteresis, but probably not unzipping. The data will be good to look at in terms of deciding on setpoints for force and velocity clamps. Just by doing these few experiments, I noticed all kinds of things which will make taking data a lot easier. One of them was how difficult it was to deal with a constant, high power beam. A quick hack may be ND filters to slide in and out of the beam after the power controller. But even better would be to use the modulation input on the controller to implement the velocity clamp. This will probably either be really easy or very hard. I need the instruction manual for that thing. I've seen it before in Evans' lab.