Koch Lab:Protocols

From OpenWetWare

Revision as of 10:55, 23 October 2009 by Anthony Salvagno (Talk | contribs)
Jump to: navigation, search
Add to My Links

This page is under construction.

Home Research Lab Members Publications Protocols Contact Funding
Principles Data Notebooks Links Meetings Presentations Inventory

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

Every lab has its own standards when it relates to lab safety, cleanliness, and shared equipment protocols. Below we've documented our specific techniques with regards to these general lab standards:

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.

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

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
Personal tools