THE ALL-PURPOSE KOCHBOOK

OF KOCHING
The American Science Classic Revised and Expanded With Over 4,500

by ANTHONY SALVAGNO and KOCHLAB

Let us give thanks
• Kochlab Nation
– Steve Koch (founder and godfather) – Larry Herskowitz – Patrick Jurney – Linh N Le – Diego Ramallo-Pardo – Nas Manole – Caleb Morse – Adam Wise (NSMS)

• The VIP List
– – – – – – Osley Lab Lopez Lab Nick Carroll Chris Lorenz Richard Yeh Karen Adelman

• The Beautiful People

Bread Making 101

• Extracting Chromatin from living yeast cells • Tethering extracted Chromatin to slide
Overview of process:
Korber et al. MCB . (2004). 24: 1096510974

Preheat the Oven to 350°

Part 1:

Part 2:

1st knead the dough…
• Need Pho5 plasmid
– Contains well documented nucleosome locations – Highly understood Nucle • To digest chromatin
us Cell W all

one needs access to nucleus
– 1st attempts involved creation of spheroplasts – Current attempts use direct contact

Mock yeast cell

…next allow dough to rise
• After extraction, tethering commences
– Chromatin needs to be prepared in a tethering construct (dig labeling on one end and biotin labeling on the other).

Above: Tethering Constuct (Steve Koch) Left: Side profile of tethered particles ready for experimentation. (Diego

Put it in the oven…
• Tethered Particle Motion
– Use “macro” microscopic reporters such as QDs or microbeads – Watch what happens

• A simple experiment: Does size matter?
– Comparing the motion of DNA when fixed to QDS and of 3 microbeads TPM
microbeads

Courtesy of Salvagno et al (2008)

… and bake for 30-40 min
• Optical Tweezers

The Tools of the Trade

• Microfluidics
– Allow automation of tethering process – Provides backbone to many future experiments

– Allow manipulation of DNA tethers

Tweezing courtesy of Dr. Koch

Microchannel by AS

Make sure to check frequently.
• Building OT
– – – – Need laser Beam steering Data Acquisition Microscope

Simple Design by Block and Neuman (2004)

Place on wire rack to cool.
• Building Microchannels
– Double Stick Tape Method – PDMS 1.0

• Using Photoresist
– Greatly improves precision of channel making – Can create all types of different shapes and sizes for use in various experiments

Mask Design by Patrick

Presentation is important
Simulations: • Thermodynamic calculations of naked DNA unzipping • Monte Carlo simulations of RNA Polymerase
– Transcription on chromatin

• Molecular Dynamics of protein-DNA interactions
– Histone-DNA and RNA Pol II-DNA – Atomistic MD for microsecond timescale – Coarse-grained MD for larger scales

More Recipes…
• Shotgun Chromatin Mapping
– Create shotgun chromatin fragments using I-PpoI – Tether fragments – Unzip fragments

…and even more!
• $6 Million (Man) Tweezers
– Beam Steering
• AOD • Mirror Galvanometer

– Better Data Acquisition – Feedback

• Micro- (and nano-) fluidic experiments
– PDMS stamping – Flow experiments

• Simulations
– Combination of atomistic and CG MD

The End