Biocurious

/ a biophysics blog

Gone fishin'

Posted 29 May 2006 by PhilipJ under

Once I’ve got labeled DNA to play with, the next step, if I’m trying to measure force-extension relations anyway, is to incubate DNA and polystyrene beads coated with the appropriate anti-body. It just so happens that the streptavidin coated beads we have are 3.17 μm in diameter, while the anti-dig beads are 2.1 μm in diameter. The larger beads better cover the tip of a micropipette, so residual suction is less of an issue. That means the 2.1 μm beads will be the ones held in the trap, and so it is these beads which I incubate with DNA.

After an hour or so the beads are covered with DNA, so I flow in with the 3.17 μm streptavidin beads, stick one on the tip of the pipette, and then flush the chamber with the DNA-covered beads. When one is trapped, I start a process we lovingly call fishing. Not entirely unlike real fishing, I move the streptavidin bead held on the micropipette away and toward the trapped bead. Normally nothing happens, but when you get a “bite”, when the biotin-labeled end of a piece of DNA finds the binding site on a streptavidin molecule, the bead being held in the trap will start getting pulled away from the trap centre, and I know I’ve got a (or perhaps many) connection.

Like real fishing, sometimes you never get a bite at all, and other days there seems to be an overabundance of them. This image is taken from one of those days when things are working too well, and while it makes for a cute image,

(and note the bead size alternating back and forth, as it has to if the labeling reaction worked as planned!), it is actually quite frustrating when this happens since I need to start all over again, flushing the chambers clean and wasting countless DNA-covered beads. Even so, these days are better than the no-bite days, without a doubt.

  

  1. Uncle Al    30 May 06    #

    From whom do you harvest the DNA, and how? Caltech had a bit of a scandal some years back with a DNA isolation lab experiment. Bring in some tissue and go for it. The cleanest most concentrated source of DNA turned out to be only directly accessible by boys. Though they were reported willing to share, quid pro quo, the girls were having none of it.


  2. PhilipJ    30 May 06    #

    The DNA used in that particular image is a plasmid harvested from hapless E. coli. The stuff I talked about in the original labeling post is λ DNA, which is commercially available in high concentration (500 µg/mL).


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