User:Steven J. Koch/Notebook/Kochlab/2009/08/05/Fork tethers
Steve Koch 12:55, 5 August 2009 (EDT): Want to make fork tethers today. I have 10 micromolar starting construct concentration, which is a huge concentration. Typically 20 picomolar is a good surface concentration. However, I don't know the actual construct concentration (it could be 1 micromolar, say). Plus, since they are shorter, I have to worry less about double-tethers, so a surface concentration of 100 or 200 pM would probably be fine. Making all of those assumptions, I'd want to dilute the construct 1:10,000. Come to think of it, maybe I should dilute the whole tube now? Nah. I'll dilute some 1:100 into BGB Pop and then dilute again before tethering.
The trick with this is that it's tough to get the surfaces to come together and form tethers when the tethering molecules are very short. I went through this at Sandia, and finally attributed it to the streptavidin microspheres being negatively charged, and if you coat the surface of the sample with DNA, it will be negatively charged too. Even small electrostatic repulsion is enough to make the beads levitate above the surface. At least this was happening to me. I'm going to go find my old notes.
- Diluting fork
- 0.5 uL 10 micromolar fork
- 250 uL 2x Pop
- 250 ul H2O
First tether sample
- Top is no DNA (6 ul sample)
- Bottom is 10 nM fork DNA (diluted above) (10 ul sample)
- antidig 5 minutes
- 5 s.v. BGB Pop; repeat for total of 3
- 1.5 s.v. DNA (or 1X pop); incubate 5 minutes
- 5 s.v. BGB Pop; repeat for total of 2 times
- 1.5 s.v beads; beads were diluted last week 1:10 into BGB Pop and sonicated (w/ Ant); incubating 10 minutes;
- washed with 5 s.v. BGB for total of 2
- sealed with nail polish
- Niether sample has many stuck beads. Fork DNA sample has maybe 1 per field of view. However, data file 23 (shotgun DNA mapping) seems to indicate unzipping to me.
- Note: when a tether broke, force did not go to "zero" but there is a big offset with the trapped bead.
- Segments 0, 1, 4, 5, 6
- There are at least 20x less stuck beads in the no DNA sample (this is good). I found two in data file 24 and neither of them ripped off. THey had very little jiggle to begin with.
- Data file 25 has a couple more that ripped off. A best guess now is that that rip off force indicates 15-20 pN. Max power was "1 watt" (as per the LCD on power controller)
- Need better tethering. Either bad DNA; or the ole short tether problem (so try tethering with higher salt concentration in beads)
Using "salt beads"
- 27 ul of the 1:10 beads from last week; 3 ul of 3.8M NaCl.
- Actually no more tethers. We (Ant and I) did find a couple loose tethers and these showed similar unzipping behavior, so I'm more convinced now that we have enough power for unzipping.
Old fork tethering notes from 2005