Koch Lab:Protocols/DNA Protocols

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Unorganized stuff

Koch Lab:Protocols/SequencesKoch Lab:Protocols/Dig-bio PCRKoch Lab:Protocols/Unzipping constructsKoch Lab:Protocols/Fork unzipping constructs

From Old Protocols Page

Labeling DNA for single-molecule stretching

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

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.

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

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

Microfluidics protocols in Lopez Keck lab/Koch lab