Koch Lab:Protocols/DNA Protocols: Difference between revisions

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==New Page==
==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/Unzipping constructs]][[Koch Lab:Protocols/Fork unzipping constructs]]
 
==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.
* [[Koch Lab:Protocols/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): [[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.
* [[Koch Lab:Protocols/Unzipping constructs|Creating dig / biotin unzipping constructs for unzipping long DNA segments]]
* [[Koch Lab:Protocols/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.
*[[Koch Lab:Protocols/Microsphere-DNA tethering|Microsphere-DNA tethering]]
*[[Koch Lab:Protocols/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
 
 
 
==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]]
 
 
 
==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]]

Latest revision as of 21:48, 8 March 2010

Needs to be organized

New Page

Unorganized stuff

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

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


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

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