Koch Lab:Protocols

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'''This page is under construction.'''
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==Protocols coming soon==
 
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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* Various methods to label dsDNA with dig and biotin for end-to-end stretching: PCR with labeled primers, klenow fill-in, ligating labeled duplexes (or hairpins), ligating multiply-tagged segments.
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==General Lab Techniques==
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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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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:
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* 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.
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*[[/How to Clean Glassware|Cleaning Glassware]]
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* Detailed protocols for "popping" experiments -- that is, unzipping DNA molecules with DNA-binding proteins present
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*[[/How to use a pipette|Using Pipettes]]
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* Various buffers used in single-molecule manipulation experiments
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*[[/Changing the Hg bulb|Changing the Hg bulb]]
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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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*[[/Laser Safety|Laser Safety]]
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===Instrumentation protocols===
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*[[/How to Autoclave|How to Autoclave]]
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* Preparing a low-tech (coverglass, slide, double-stick stape) sample chamber
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*[[/How to Clean the Autoclave|How to Clean the Autoclave]]
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* Flow cells for electromagnetic steering of microtubules labeled with magnetic microspheres.
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*[[/Recycling|Recycling]]
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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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* Making a flow cell to hydrate a SUMMiT MEMS device
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==Molecular Biology Protocols==
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===Computational protocols===
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===Standard Techniques===
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* 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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*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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===KochLab Techniques===
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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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==Equipment Setup==
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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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==Computational Protocols==
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*Shotgun DNA Mapping
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*Kinesin Processivity
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*Kinesin Tracking
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*Motion Detection Camera
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*[[/Loading rate clamp|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.
* 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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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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