OpenWetWare - Koch Lab

Koch Lab:Protocols/Kinesin/BRB80 Acid Pipes

2013-03-21T00:42:02Z

Various BRB80 buffers can be found on the kinesin home page [1].

* Below is a version specific for potassium ions (as opposed to sodium). The Google Doc can be found here: http://spreadsheets.google.com/pub?key=pYVXjfJA8GQt0djdIdn6_Tw [2]
* (Steve Koch [3]:I can't remember whether I made this buffer for use with malachite green inorganic phosphate assays, or because I thought it was better to use potassium ions versus sodium ions in kinesin assays.)

View/Edit Spreadsheet [4]

Koch Lab:Protocols/Kinesin/BRB80 Acid Pipes

2013-03-21T00:42:02Z

Koch Lab:Protocols/Changing the Hg bulb

2011-06-09T22:18:34Z

The below steps outline how to remove and properly replace a 100W Hg bulb in an Olympus Hg bulb housing. Make sure to wear gloves as any oils on the bulb can cause it to explode. Also, make sure not to touch the bulb as anything that can burn on it will burn and can cause it to explode.

Once the bulb has been replaced, make sure to run it for at least an hour in order to make sure that the plasma burns in a preferential spot in the electrodes.

STEP 1

Remove the Hg lamp from the microscope. First, take out the iris lever.

[1]

Next, loosen the set screws holding the lamp in place.

[2]

Remove the lamp carefully making sure to include the ND filter sets with removal.

[3]

Set the lamp on the table and point it to the wall.

[4]

STEP 2

Next, unscrew

Koch Lab:People

2011-06-09T21:35:10Z

[1]

Home [2]
Research [3]
LAB MEMBERS
Publications [4]
Protocols [5]
Contact [6]
Funding [7]

Principles [8]
Data [9]
Notebooks [10]
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

Use This Link To See Recent Wiki Editing By Kochlab Members And Alums [15]

CURRENT OR RECENT MEMBERS

[16]

Steve Koch [17], Assistant Professor, Physics,
Open Notebook [18] [19]

Anthony Salvagno [20], Graduate Student, Physics,
Open Notebook [21] [22]

Brian P. Josey [23], Undergraduate Student, Physics [24]

Pranav Rathi My Project page [25], Optics Ph.D. student

[26]

Nadia Fernandez-Oropeza [27], Graduate Student, Physics [28]

Priyanka Rawat, Visiting scientist [29]

Kenji Doering [30], 2011 Summer REU Student

==

Koch Lab:Publications

2011-06-09T17:47:09Z

[1]

Home [2]
Research [3]
Lab Members [4]
PUBLICATIONS
Protocols [5]
Contact [6]
Funding [7]

Principles [8]
Data [9]
Notebooks [10]
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

KINESIN / MICROTUBULE MOLECULAR MOTOR SYSTEM

EFFECTS OF CASEIN SURFACE PASSIVATION IN KINESIN-1 GLIDING MOTILITY ASSAY

[15] [16]

Maloney A, Herskowitz LJ, Koch SJ (2011) Effects of Surface Passivation on Gliding Motility Assays. PLoS ONE 6(6): e19522. http://dx.doi.org/10.1371/journal.pone.0019522 [17]

Andy Maloney studies the effects of alpha, beta, kappa, and whole casein in the kinesin-1 gliding motility assay. Automated speed analysis software by Larry Herskowitz.
Figure: Black circles are alpha casein passivation, red squares are beta casein passivation, green up pointing triangles are whole casein passivation, and blue down pointing triangles are mixed casein passivation. Each data point is the mean from three different samples, taken at approximately the same assay time. Error bars represent the standard error of the mean. Alpha casein had

Koch Lab:Protocols/Sequences

2011-02-24T15:20:34Z

OLIGONUCLEOTIDES

* Unzipping adapter and hairpin oligos [1]
* Unzipping fork oligos (dig & biotin labeled for direct tethering) [2]
* PCR Primers [3]

PLASMIDS

* pRL574 [4]
* DNA 2.0 custom plasmid - pALS [5]

OTHER DNA: GENOME FRAGMENTS, ETC.

* pBR322 unzipping construct from 2002 [6]
* pCP681 unzipping construct from 2002 [7]
* All Anthony\'s DNA Sequences [8]

Koch Lab:Protocols/Sequences

2011-02-24T15:20:34Z

Koch Lab:Protocols

2011-01-24T18:40:30Z

THIS PAGE IS UNDER CONSTRUCTION. [1]

Home [2]
Research [3]
Lab Members [4]
Publications [5]
PROTOCOLS
Contact [6]
Funding [7]

Principles [8]
Data [9]
Notebooks [10]
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

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 [15] 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.

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:

* Cleaning Glassware [16]
* [[Koch Lab:Protocols/How to use a pipette|Using Pipettes]

Koch Lab:Meetings/Symposium

2009-05-21T21:59:58Z

SCHEDULE OF TALKS

View/Edit Spreadsheet [1]

LUNCH OPTIONS

* Relish

* Chinese

* Thai House
* Kai's

* Hispanic Cultural Center
* Pizza
* Nick's

LIST OF TALKS

* Ant's first talk: Media:Biological things you should know (51509).pptx [2]
* Ant's second talk: Media:The Biological Preparation of Shotgun DNA Mapping (51509).pptx [3]
* IK's afternoon talk: On osmotic stress and protein dissociation rates (pdf) [4]

Koch Lab:Data/MT Gliding Assay Readme File

2010-11-18T19:30:15Z

This is the readme file for kochlab gliding assay and microtubule tracking data.
[1]

[2]
To the extent possible under law, Steven J. Koch and other KochLab members [3] has waived all copyright and related or neighboring rights to KochLab Data. This work is published from

Koch Lab:Data/MT Gliding Assay Readme File

2010-11-18T19:30:15Z

Koch Lab:Notebooks

2010-09-17T20:00:37Z

[1]

Home [2]
Research [3]
Lab Members [4]
Publications [5]
Protocols [6]
Contact [7]
Funding [8]

Principles [9]
Data [10]
NOTEBOOKS
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

CONTENTS

* 1 Use this link to see recent wiki editing by Kochlab members and alums [15]

* 2 KochLab members\' public notebooks [16]

* 2.1 Archived [17]

* 3 Recent changes by KochLab members [18]

Use This Link To See Recent Wiki Editing By Kochlab Members And Alums [19]

KOCHLAB MEMBERS\' PUBLIC NOTEBOOKS

* Anthony Salvagno [20]
* Andy Maloney [21]
* Larry Herskowitz [22]
* Brian Josey [23]
* Pranav Rathi [24]
* Nadia Fernandez-Oropeza [25]
* Steve Koch [26]

ARCHIVED

* Brigette Black [27]
* Caleb Morse [28]
* Linh Le [29]
* Emmalee Jones [30]

RECENT CHANGES BY KOCHLAB MEMBERS

{{Special:Recentchanges/u=Steven J. K

Koch Lab:Presentations/Steve's Presentations

2010-09-08T05:25:41Z

This is a page that highlights all of Steve's public talks and some for a closed audience. The oldest presentations are at the bottom. For other presentations not included on this page go to:
Steve\'s Slideshare [1] or Steve on Nature Precedings [2]

NOVEMBER 5, 2009 OVERVIEW OF PROTEINS IN HEAVY WATER

2009 November Heavy Water Update [3]

Koch Lab:Presentations

2010-09-08T05:24:59Z

[1]

Home [2]
Research [3]
Lab Members [4]
Publications [5]
Protocols [6]
Contact [7]
Funding [8]

Principles [9]
Data [10]
Notebooks [11]
Links [12]
Meetings [13]
PRESENTATIONS
Inventory [14]

As of September 2010, We're using Nature Precedings and Slideshare to share most of our presentations. Here's some links:

* Steve Koch on Nature Precedings [15]
* Steve Koch on Slideshare [16] and scribd [17]
* Anthony Salvagno on Slideshare [18] and scribd [19]

PEOPLE\'S PRESENTATIONS

(These pages are falling out of date)
STEVE\\'S PRESENTATIONS [20]
ANTHONY\\'S PRESENTATIONS [21]
LARRY\\'S PRESENTATIONS [22]
ANDY\\'S PRESENTATIONS [23]

Koch Lab:Funding

2010-09-08T05:20:23Z

[1]

Home [2]
Research [3]
Lab Members [4]
Publications [5]
Protocols [6]
Contact [7]
FUNDING

Principles [8]
Data [9]
Notebooks [10]
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

ACTIVE OR RECENT FUNDING

[15]

DEFENSE THREAT REDUCTION AGENCY [16]

March 2009-March 2012, HDTRAI-09-1-0018, $703,830 total for KochLab component of project.
Title: Coupled Atomistic Modeling and Experimental Studies of Energy Transduction and Catalysis in the Molecular Motor Protein Kinesin
Role: Co-PI (PI: Susan Atlas, University of New Mexico)

[17]

ADDGENE RESOURCE SHARING AWARD [18]

September 2010, $5,000 gift to Andy Maloney [19] and our lab in recognition of open science contributions.
Role: PI, but Andy Maloney did all the work for the application.

PRIOR FUNDING

[20]

CENTER FOR HIGH TECHNOLOGY MATERIALS [21]

Start-up funds for our lab

Koch Lab:Protocols/Optical Tweezers

2010-07-27T22:36:54Z

Userboxes [1]

[2]
This equipment incorporates components from Mad City Labs [3]

[4]
This equipment incorporates components from ThorLabs [5]

[6]
This equipment incorporates components from Newport [7]

-------------------------
[8]

CONTENTS

* 1 Background [9]

* 2 Setup [10]

* 2.1 Basic Setup [11]
* 2.2 Our Setup [12]

BACKGROUND

SETUP

Optical Tweezers are simple in theory but are not very simple in setup. This is why we are going to detail our setup so that others may replicate. The other reason for this section is because different people build different tweezers for different applications. We require one tweezer with sample control coming from stage movement. Other labs use bi-directional tweezers (clamping from 2 sides), multiple tweezers, advanced tweezer positioning, and more.

BASIC SETUP

The simplest form of OT is laser light shined through an objective. Of course with this simple setup nothing can be achieved. All you have is a trap. There is no way to detect what is happening in the trap. There is no way to move the trap. This leads to more complex designs determined from experimental need.

OUR SETUP

We have some requirements based on the types of

Koch Lab:Protocols/Recycling

2010-06-30T20:54:26Z

We have a new system for recycling all of our plastics, paper and waste. So we have rearranged the collection bins as such: LARGE BIN UNDER THE AUTOCLAVE- This bin is for the tips and tubes that we throw out. Make sure that everything is empty before you dispose of it in the container. ROUND BIN NEAR THE MAIN DOOR- This large bin for general recycling, like plastic soda bottles or cans. The tip holders from the manufacturer also go into this bin. CARDBOARD BOX ON TOOL BOX- This box is
still used for the recycling of paper. SMALL TRASH CANS- The small bins all over the place are for trash in general.

Koch Lab:Notebooks/Brian Joseys Notebook

2009-08-25T18:28:59Z

[1]

Search this Project

Customize your entry pages [2] [3]

PROJECT DESCRIPTION/ABSTRACT

* Place short description of project or notes regarding this project

* Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Donec porta commodo tellus. Nam a est eget libero mollis tincidunt. Aliquam purus. Quisque nulla ligula, facilisis in, pulv

Koch Lab:Notebook/Kinesin Project Summer09

2009-06-01T19:37:54Z

Koch Lab

2010-06-02T20:38:06Z

[1] [2]

HOME
Research [3]
Lab Members [4]
Publications [5]
Protocols [6]
Contact [7]
Funding [8]

Principles [9]
Data [10]
Notebooks [11]
Links [12]
Meetings [13]
Presentations [14]
Inventory [15]

WELCOME TO THE KOCHLAB WIKI!

We are a research lab at the University of New Mexico [16], in the Center for High Technology Materials (CHTM) [17] and Physics Department [18]. We are experimental biophysicists, which to us means we use our skills and talents as physical scientists to provide new answers for important questions in cellular and molecular biology. For example, developing new techniques to probe protein-DNA interactions by unzipping single DNA molecules with optical tweezers. You can learn more about our
interests on our Research [19] page.

In addition, we are committed to being open scientists and sharing our results, methods, and ideas as quickly, clearly, and openly as possible. However, being a very new lab, we are still learning how to do this and are

Koch Lab

2010-06-02T20:38:06Z

Koch Lab

2010-06-02T20:38:06Z

Koch Lab:Data

2010-05-13T22:56:37Z

[1]

Home [2]
Research [3]
Lab Members [4]
Publications [5]
Protocols [6]
Contact [7]
Funding [8]

Principles [9]
DATA
Notebooks [10]
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

As much as possible, we're going to share all of our data publicly. To make it useful, we're also going to post our data analysis code and instructions for how to analyze data. This is a work in progress, though, and so far all we can post are links to lots of data and some notes about the data. Please contact us if you want to know how to use the data!

KOCHLAB DATA

[15]

[16]
To the extent possible under law,

Koch Lab:Publications/Drafts/Versatile Feedback/Paper

2010-03-16T20:53:34Z

CONTENTS

* 1 Link to Nature Precedings Version [1]
* 2 Title [2]
* 3 Authors [3]
* 4 Abstract [4]

LINK TO NATURE PRECEDINGS VERSION

* Nature Precedings version [5].

TITLE

Versatile Control System for Automated Single-Molecule Optical Tweezers Investigations

AUTHORS

Richard C. Yeh [6] and Steven J. Koch [7]

ABSTRACT

We present a versatile control system to automate single-molecule biophysics experiments. This method combines low-level controls into various functional, user-configurable modules, which can be scripted in a rudimentary instruction language. The ease with which the high-level parameters can be changed accelerates the development of a durable experiment for the perishable single-molecule samples. Once the experimental parameters are tuned, the control system can be used to repeatedly
manipulate other single molecules in the same way, which is necessary to accumulate the statistics needed to report results fr

Koch Lab:Publications/Drafts/Versatile Feedback

2010-03-16T20:52:47Z

This is a draft of a paper that describes a useful software controller design for automated experimental setups. The particular application is dynamic single-molecule experiments performed with optical traps, but we believe the abstraction of hardware control into modular, functional feedback steps with user-programmable stop conditions offers a good example that can have application in other fields.

The draft was mostly written by Richard Yeh, with some help from Steve Koch. We are posting on OpenWetWare in draft form, because we probably will not try to publish in a peer reviewed journal soon, and the draft as is may have some positive impact.

* Text of the paper [1]

* Nature Precedings Version [2]

* Why you should use our software [3]
* /Software [4]

Koch Lab:Research

2010-03-10T15:24:07Z

[1]

Home [2]
RESEARCH
Lab Members [3]
Publications [4]
Protocols [5]
Contact [6]
Funding [7]

Principles [8]
Data [9]
Notebooks [10]
Links [11]
Meetings [12]
Presentations [13]
Inventory [14]

OPEN-RESEARCH PROJECTS

* /MediaWiki for Open Science [15]
* How to build your own laser diode [16]

ANTHONY\\'S LAB NOTEBOOK [17]

In his notebook, Anthony covers a wide range of topics including:

* Optical tweezers construction

* Molecular Biology protocols and experiments

* DNA ligation

* Unzipping Experiments

SOME OF THE BIOLOGICAL PROBLEMS WE ARE ACTIVELY INTERESTED IN

* The molecular motor kinesin [18]. We have a collaborative project with the Susan Atlas theory group to study the mechanochemistry of kinesin-1. This project is funded by a 3-year DTRA basic research grant. From the experimental side, we are investigation how water affects the mechanochemistry and stepping process. We have found two new avenues to explore: osmotic stress and isotope effects (e.g. deuterium versus hy

Koch Lab:Protocols/Flow cell

2010-03-09T15:37:53Z

KINESIN TECHNIQUE

Kinesin assays can be very frustrating when things go wrong. There are many steps in the preparation and any one of those steps can cause the assay to not work if improperly executed. Since there are so many steps, the experimenter is left in the dark when things go wrong since it is the final product that we look at and none of the steps in between. In order to prevent inadvertent mistakes from occurring when making flow cells, this page is going to outline how to make them. This flow cell is
specific for kinesin assays however, the techniques can apply to preparation of other flow cells for other experiments.

SLIPS & SLIDES

We use slides [1] (#48300-025) and slips [2] (#48366-045) purchased from VWR, pictured below.

[3]

=

Koch Lab:Protocols/DNA Protocols

2010-03-09T04:48:42Z

Needs to be organized

CONTENTS

* 1 New Page [1]
* 2 Unorganized stuff [2]

* 3 From Old Protocols Page [3]

* 3.1 Labeling DNA for single-molecule stretching [4]
* 3.2 Labeling DNA for unzipping [5]

NEW PAGE

UNORGANIZED STUFF

Koch Lab:Protocols/Sequences [6]Koch Lab:Protocols/Dig-bio PCR [7]Koch Lab:Protocols/Unzipping constructs [8]Koch Lab:Protocols/Fork unzipping constructs [9]

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 [10]
* 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 [11]

LABELING DNA FOR UNZIPPING

More complicated construction of a molecules that will unzip when stretched, as in our 2002 Biophys. J. paper PMID 12124289 [12]. 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|Creat

Koch Lab:Publications/Supplemental info for MEMS force sensor gravitational calibration

2010-03-08T22:20:05Z

SUPPLEMENTAL INFORMATION FOR "MICROMACHINED PICONEWTON FORCE SENSOR FOR BIOPHYSICS INVESTIGATIONS"

Steve Koch [1] 16:58, 8 March 2010 (EST): We developed and used a gravitational-based calibration method to calibrate the spring used in the APL report [2]. At the time (2006), we expected to pursue the work a bit further and write up a methods paper describing it further. However, all of the co-authors on the paper left their groups at Sandia shortly thereafter, and we were unable to take further data. To at least have a small benefit, I'm going to post the details of the method here.

Information for original publication:

* Micromachined piconewton force sensor for biophysics investigations
* Steven J. Koch, Gayle E. Thayer, Alex D. Corwin, and Maarten P. de Boer
* Appl. Phys. Lett. 89, 173901 (2006); doi:10.1063/1.2364118 (3 pages) [3]

==Description of method for calibrating spring c

Koch Lab:Protocols/Microsphere-DNA tethering/Glass, dig, biotin, microsphere, 4kb DNA/Cleaning glass

2010-03-05T16:03:24Z

Even without cleaning the glass surfaces, we have been able to achieve decent tethering (high tether density relative to stuck beads) following the protocol on the parent page [1]. However, a standard and repeatable cleaning method is bound to provide more consistent results and is likely desirable (although, the cleaning procedure may not necessarily increase the tether density, I can't promise that). There are several different methods we are familiar with, and others we are not. In order of
familiarity below:

CRAZY WINDEX METHOD

In graduate school, Steve [2] mostly desired consistent results. He reasoned that Windex was good at making glass appear to be clean, and is cheap. He also reasoned that residue left behind by the Windex was likely more consistent than whatever gunk is on