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/Unzipping constructs]][[Koch Lab:Protocols/Fork unzipping constructs | [[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== | ==From Old Protocols Page== | ||
===Labeling DNA for single-molecule stretching=== | ===Labeling DNA for single-molecule stretching=== | ||
Various methods to label dsDNA with digoxigenin (dig) and biotin for end-to-end stretching. | Various methods to label dsDNA with digoxigenin (dig) and biotin for end-to-end stretching. | ||
* [[/Dig-bio PCR|PCR with labeled primers]] | * [[Koch Lab:Protocols/Dig-bio PCR|PCR with labeled primers]] | ||
* klenow fill-in | * klenow fill-in | ||
* ligating labeled duplexes (or hairpins) | * ligating labeled duplexes (or hairpins) | ||
* ligating multiply-tagged segments. | * ligating multiply-tagged segments. | ||
** (Useful for a couple of the above protocols): [[Koch Lab:Protocols/Oligonucleotide Annealing]] | ** (Useful for a couple of the above protocols): [[Koch Lab:Protocols/Oligonucleotide Annealing]] | ||
===Labeling DNA for unzipping=== | ===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. | 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]] | * [[Koch Lab:Protocols/Unzipping constructs|Creating dig / biotin unzipping constructs for unzipping long DNA segments]] | ||
* [[/Fork unzipping constructs|Hybridized unzipping forks]] | * [[Koch Lab:Protocols/Fork unzipping constructs|Hybridized unzipping forks]] | ||
===DNA tethering=== | ===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. | 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]] | *[[Koch Lab:Protocols/Microsphere-DNA tethering|Microsphere-DNA tethering]] | ||
*[[/Glass-DNA-PDMS tethering|Glass-DNA-PDMS tethering]] | *[[Koch Lab:Protocols/Glass-DNA-PDMS tethering|Glass-DNA-PDMS tethering]] | ||
===Probing protein-DNA interactions by unzipping single DNA molecules=== | ===Probing protein-DNA interactions by unzipping single DNA molecules=== | ||
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.
- 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.
- 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
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
- For specific protocol see the following link
- Photo lithography
- PDMS
