Koch Lab:Protocols/DNA Protocols

Needs to be organized

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

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.
 * (Useful for a couple of the above protocols): Koch Lab:Protocols/Oligonucleotide Annealing

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.
 * Creating dig / biotin unzipping constructs for unzipping long DNA segments
 * Hybridized unzipping forks

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

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

 * For specific protocol see the following link
 * Photo lithography
 * PDMS