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.
General Lab Techniques
Molecular Biology Protocols
Standard Techniques
- PCR
- Gel Electrophoresis
- Cloning
- Transformation into E. Coli
KochLab Techniques
Equipment Setup
Computational Protocols
- Shotgun DNA Mapping
- Kinesin Processivity
- Kinesin Tracking
- Motion Detection Camera
- Loading Rate Clamp
Old Page to be deleted
Everything below this point will be deleted by Friday Oct 23. If you have something recorded below that you want saved, please move information to the appropriate location above. All of the wiki links below should be under the DNA Protocols above as of now, but if anything is left out please contribute.
DNA Sequences
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.
- (Useful for a couple of the above protocols): Oligo 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.
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)
Microfluidics protocols in Lopez Keck lab/Koch lab
- For specific protocol see the following link
- Photo lithography
- PDMS
