Koch Lab:Protocols

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One of the goals of our lab is to share protocols in a form that makes it as easy as possible for other labs to build off of them. These will be a combination of protocols that we will develop and also those that Steve has developed in the past but has not had a good opportunity to distribute yet. If any of the following protocols are of particular interest, drop us a line or make a note on the discussion page! We also want to share software applications and computational modules (mostly LabVIEW) that would be useful to others.

Molecular protocols

Labeling DNA for single-molecule stretching

Various methods to label dsDNA with digoxigenin (dig) and biotin for end-to-end stretching.

• PCR with labeled primers
• klenow fill-in
• ligating labeled duplexes (or hairpins)
• ligating multiply-tagged segments.

Labeling DNA for unzipping

More complicated construction of a molecules that will unzip when stretched, as in our 2002 Biophys. J. paper PMID 12124289. Compared with DNA stretching, making a construct for unzipping presents many more potential pitfalls, and it is also challenging to get good yield.

DNA tethering

Making single-molecule tethers via antidig-dig and biotin-streptavidin. Including all the tricks for washing glass, blocking, how much DNA to use, microsphere selection, microsphere preparation, etc.

• Microsphere-DNA tethering
• Glass-DNA-PDMS tethering

Probing protein-DNA interactions by unzipping single DNA molecules

Detailed protocols for "popping" experiments -- that is, unzipping DNA molecules with DNA-binding proteins present

Single-molecule manipulation buffers

Various buffers used in single-molecule manipulation experiments

Kinesin aggregation via DLS

Measuring kinesin aggregation via dynamic light scattering (DLS) (As in our kinesin paper)

Instrumentation protocols

• Preparing a low-tech (coverglass, slide, double-stick stape) sample chamber
• Flow cells for electromagnetic steering of microtubules labeled with magnetic microspheres.
• Placing single 3 micron magnetic microspheres (or also 30 micron polystyrene) onto MEMS devices (with micromanipulators) as in 2006 Appl. Phys. Let. (PDF)
• Making a flow cell to hydrate a SUMMiT MEMS device
• Some things about AODs

Computational protocols

• Loading rate clamp--method for stretching single-molecule tethers such that the force-versus-time curves are linear segments. Used in 2003 Phys. Rev. Let. paper, PMID 12906513, for unzipping with protein present, but can be used whenever the polymer physics are known ahead of time.
• Maximum likelihood anlaysis of single-molecule disruption data with Evan Evans' et al. Dynamic Force Spectroscopy (DFS) model. Much better and easier than performing Gaussian fits to histograms. Used in 2003 Phys. Rev. Let. paper, PMID 12906513.
• Finite Element Magnetic Modeling (FEMM) for predicting forces on magnetic microspheres, as in 2006 Appl. Phys. Let. (PDF)

Photo lithography protocol in Keck lab/Koch lab

• For specific protocol see the following link
• Photo lithography