1.
THE ALL-PURPOSE KOCHBOOK
OF
KOCHING
The American Science Classic
Revised and Expanded
With Over 4,500 Experiments
And 1,000 Informative Illustrations
by ANTHONY SALVAGNO
and KOCHLAB

2.
Let us give thanks
• Kochlab Nation • The VIP List
– Steve Koch (founder and – Osley Lab
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)

3.
Bread Making 101
Biological
Methods
•Chromatin
Physical Methods Extraction
•DNA Molecule
•Optical Tweezers
Tethering
•Microfluidics
•Nanomanipulation
(future)
Modeling
Methods
•Molecular Dynamics
•Monte Carlo
Simulations
Better Understanding of
Cancer on the “Nano” Scale

4.
Preheat the Oven to 350°
• Extracting Chromatin from living yeast cells
• Tethering extracted Chromatin to slide
Korber et al. MCB . (2004). 24: 10965-10974
Overview of process:
Part 1: Part 2:

5.
1st knead the dough…
• Need Pho5 plasmid
– Contains well documented nucleosome locations
– Highly understood
• To digest chromatin one
Nucleus Cell
Wall
needs access to nucleus
– 1st attempts involved
creation of spheroplasts
– Current attempts use
Mock yeast cell direct contact with
From Wikimedia Commons
nucleus

6.
…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
Ramallo-Pardo)

7.
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)

8.
… and bake for 30-40 min
The Tools of the Trade
• Optical Tweezers • Microfluidics
– Allow manipulation of – Allow automation of
DNA tethers tethering process
– Provides backbone to
many future experiments
Tweezing courtesy of Dr. Koch Microchannel by AS

9.
Make sure to check frequently.
• Building OT
– Need laser
– Beam steering
– Data Acquisition
– Microscope
Simple Design by Block and Neuman (2004)

10.
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 Jurney

11.
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

12.
More Recipes…
• Shotgun Chromatin Mapping
– Create shotgun chromatin
fragments using I-PpoI
– Tether fragments
– Unzip fragments

13.
…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

14.
The End