Difference between revisions of "Koch Lab:Protocols"

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(KochLab Techniques)
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*[[/Loading rate clamp|Loading Rate Clamp]]
*[[/Loading rate clamp|Loading Rate Clamp]]
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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.
* [[/Dig-bio PCR|PCR with labeled primers]]
* klenow fill-in
* ligating labeled duplexes (or hairpins)
* ligating multiply-tagged segments.
** (Useful for a couple of the above protocols): [[/Oligonucleotide Annealing|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.
* [[/Unzipping constructs|Creating dig / biotin unzipping constructs for unzipping long DNA segments]]
* [[/Fork unzipping constructs|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|Microsphere-DNA tethering]]
*[[/Glass-DNA-PDMS 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 [http://dx.doi.org/10.1016/j.fgb.2007.02.004 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 [http://link.aip.org/link/?APL/89/173901 2006 Appl. Phys. Let.] ([http://www.chtm.unm.edu/publications/APL%2089_173901_Koch,%20Thayer,%20Corwin,%20de%20Boer_MEMS%20force%20sensor%20for%20mag%20bead%20calibration.pdf PDF])
* Making a flow cell to hydrate a SUMMiT MEMS device
* [[Koch Lab:Research/AOD tidbits|Some things about AODs]]

Revision as of 14:13, 26 October 2009

This page is under construction.

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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

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