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 • The VIP List
– Steve Koch (founder – Osley Lab
and godfather) – Lopez Lab
– Larry Herskowitz – Nick Carroll
– Patrick Jurney – Chris Lorenz
– Linh N Le – Richard Yeh
– Diego Ramallo-Pardo – Karen Adelman
– Nas Manole • The Beautiful People
– Caleb Morse
– Adam Wise (NSMS)
Bread Making 101
 Preheat the Oven to
350°
• Extracting Chromatin from living
yeast cells
• Tethering extracted Chromatin to
slide

Korber et al. MCB . (2004). 24: 10965-

Overview of 10974
Part process: Part
1: 2:
 1st knead the dough…
• Need Pho5 plasmid
– Contains well documented nucleosome
locations
Nucle
– Highly understood
• To digest chromatin
Cell
us W
all one needs access
to nucleus
– 1st attempts
involved creation of
Mock yeast spheroplasts
cell
– Current attempts
use direct contact
 …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 microbeads
TPM of 3
microbeads

Courtesy of Salvagno
et al (2008)
 … and bake for 30-40 min
The Tools of the
Trade
• Optical Tweezers • Microfluidics
– Allow manipulation – Allow automation of
of DNA tethers tethering process
– Provides backbone
to many future
experiments

Tweezing courtesy of Microchannel

Dr. Koch 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