Koch Lab:Protocols/DNA Protocols: Difference between revisions

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==Unorganized stuff==
==Unorganized stuff==
[[Koch Lab:Protocols/Sequences]][[Koch Lab:Protocols/Dig-bio PCR]][[Koch Lab:Protocols/Oligonucleotide Annealing]][[Koch Lab:Protocols/Unzipping constructs]][[Koch Lab:Protocols/Fork unzipping constructs]][[Koch Lab:Protocols/Microsphere-DNA tethering]][[Koch Lab:Protocols/Photolithography]][[Koch Lab:Protocols/PDMS]]
[[Koch Lab:Protocols/Sequences]][[Koch Lab:Protocols/Dig-bio PCR]][[Koch Lab:Protocols/Oligonucleotide Annealing]][[Koch Lab:Protocols/Unzipping constructs]][[Koch Lab:Protocols/Fork unzipping constructs]][[Koch Lab:Protocols/Microsphere-DNA tethering]][[Koch Lab:Protocols/Photolithography]][[Koch Lab:Protocols/PDMS]]
==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.
* [[/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]]
==Computational protocols==
* [[/Loading rate clamp|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 [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])
==Microfluidics protocols in Lopez Keck lab/Koch lab==
*For specific protocol see the following link
*[[Koch Lab:Protocols/Photo lithography|Photo lithography]]
*[[Koch Lab:Protocols/PDMS|PDMS]]

Revision as of 13:20, 22 October 2009

Needs to be organized

Unorganized stuff

Koch Lab:Protocols/SequencesKoch Lab:Protocols/Dig-bio PCRKoch Lab:Protocols/Oligonucleotide AnnealingKoch Lab:Protocols/Unzipping constructsKoch Lab:Protocols/Fork unzipping constructsKoch Lab:Protocols/Microsphere-DNA tetheringKoch Lab:Protocols/PhotolithographyKoch Lab:Protocols/PDMS

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

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

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