Koch Lab:Protocols

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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, [[Koch_Lab:Contact|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.
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, [[Koch_Lab:Contact|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.
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==Molecular protocols==
 
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===Labeling DNA for single-molecule stretching===
 
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Various methods to label dsDNA with digoxigenin (dig) and biotin for end-to-end stretching.
 
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* [[/Dig-bio PCR|PCR with labeled primers]]
 
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* klenow fill-in
 
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* ligating labeled duplexes (or hairpins)
 
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* ligating multiply-tagged segments.
 
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** (Useful for a couple of the above protocols): [[/Oligonucleotide Annealing|Oligo Annealing]]
 
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===Labeling DNA for unzipping===
 
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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.
 
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* [[/Unzipping constructs|Creating dig / biotin unzipping constructs]]
 
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===DNA tethering===
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==General Lab Techniques==
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Making single-molecule tethers via antidig-dig and biotin-streptavidinIncluding all the tricks for washing glass, blocking, how much DNA to use, microsphere selection, microsphere preparation, etc.
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Every lab has its own standards when it relates to lab safety, cleanliness, and shared equipment protocolsBelow we've documented our specific techniques with regards to these general lab standards:
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*[[/Microsphere-DNA tethering|Microsphere-DNA tethering]]
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*[[/How to Clean Glassware|Cleaning Glassware]]
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*[[/Glass-DNA-PDMS tethering|Glass-DNA-PDMS tethering]]
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*[[/How to use a pipette|Using Pipettes]]
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*[[/Changing the Hg bulb|Changing the Hg bulb]]
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*[[/Laser Safety|Laser Safety]]
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*[[/How to Autoclave|How to Autoclave]]
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*[[/How to Clean the Autoclave|How to Clean the Autoclave]]
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*[[/Recycling|Recycling]]
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===Probing protein-DNA interactions by unzipping single DNA molecules===
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==Molecular Biology Protocols==
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Detailed protocols for "popping" experiments -- that is, unzipping DNA molecules with DNA-binding proteins present
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===Standard Techniques===
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*PCR
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*[[User:Anthony Salvagno/Notebook/Research/Notes About Gels|Gel Electrophoresis]]
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*Cloning
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*Transformation into E. Coli
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===Single-molecule manipulation buffers===
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===KochLab Techniques===
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Various buffers used in single-molecule manipulation experiments
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*[[/Kinesin|Kinesin protocols]]
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*[[/DNA Protocols|DNA Protocols]]
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*[[/Flow cell|Flow cell]]
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===Kinesin aggregation via DLS===
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==Equipment Setup==
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Measuring kinesin aggregation via dynamic light scattering (DLS) (As in our [http://dx.doi.org/10.1016/j.fgb.2007.02.004 kinesin paper])
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*[[/Optical Tweezers|Optical Tweezers]]
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*[[/Microscopy|Microscopy]]
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*[[/Gel Imaging System|Gel Imaging System]]
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==Instrumentation protocols==
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==Computational Protocols==
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* Preparing a low-tech (coverglass, slide, double-stick stape) sample chamber
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*Shotgun DNA Mapping
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* Flow cells for electromagnetic steering of microtubules labeled with magnetic microspheres.
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*Kinesin Processivity
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* 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])
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*Kinesin Tracking
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* Making a flow cell to hydrate a SUMMiT MEMS device
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*Motion Detection Camera
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* [[Koch Lab:Research/AOD tidbits|Some things about AODs]]
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*[[/Loading rate clamp|Loading Rate Clamp]]
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==Computational protocols==
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* [[/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.
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* 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.
* 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])
* 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])
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==Microfluidics protocols in Lopez Keck lab/Koch lab==
 
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*For specific protocol see the following link
 
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*[[Koch Lab:Protocols/Photo lithography|Photo lithography]]
 
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*[[Koch Lab:Protocols/PDMS|PDMS]]
 
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Current revision

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.

Contents

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

KochLab Techniques

Equipment Setup

Computational Protocols

  • Shotgun DNA Mapping
  • Kinesin Processivity
  • Kinesin Tracking
  • Motion Detection Camera
  • Loading Rate Clamp
  • 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)


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