THE ALL-PURPOSE KOCHBOOK




      OF
      KOCHING
        The American Science Classic
        Revised and Expanded
    ...
Let us give thanks
• Kochlab Nation               • The VIP List
   – Steve Koch (founder and      –   Osley Lab
     godf...
Bread Making 101

                               Biological
                               Methods
                       ...
Preheat the Oven to 350°
  • Extracting Chromatin from living yeast cells
  • Tethering extracted Chromatin to slide




 ...
1st knead the dough…
 • Need Pho5 plasmid
       – Contains well documented nucleosome locations
       – Highly understoo...
…next allow dough to rise
• After extraction, tethering commences
  – Chromatin needs to be prepared in a tethering
    co...
Put it in the oven…
• Tethered Particle Motion
   – Use “macro” microscopic reporters such as QDs or microbeads
   – Watch...
… and bake for 30-40 min
                        The Tools of the Trade
• Optical Tweezers                  • Microfluidic...
Make sure to check frequently.
• Building OT
  –   Need laser
  –   Beam steering
  –   Data Acquisition
  –   Microscope
...
Place on wire rack to cool.
• Building Microchannels
   – Double Stick Tape Method
   – PDMS 1.0
• Using Photoresist
   – ...
Presentation is important
Simulations:
• Thermodynamic calculations of naked DNA
  unzipping
• Monte Carlo simulations of ...
More Recipes…
  • Shotgun Chromatin Mapping
    – Create shotgun chromatin
      fragments using I-PpoI
    – Tether fragm...
…and even more!
• $6 Million (Man) Tweezers
  – Beam Steering
     • AOD
     • Mirror Galvanometer
  – Better Data Acquis...
The End
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The Joy Of Koching 6/13/08

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This was a practice talk I gave in front of the IGERT people at UNM. It is a brief discussion of the things I do in KochLab up to this point (Summer 2008).

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  • Put up some cool pictures
  • Qd: about 0.02± .003 μm2Microsphere: 0.05± .006 μm2
  • Need pics from mac and need pic of pdms 1.0
  • The Joy Of Koching 6/13/08

    1. 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. 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. 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. 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. 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. 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. 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. 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. 9. Make sure to check frequently. • Building OT – Need laser – Beam steering – Data Acquisition – Microscope Simple Design by Block and Neuman (2004)
    10. 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. 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. 12. More Recipes… • Shotgun Chromatin Mapping – Create shotgun chromatin fragments using I-PpoI – Tether fragments – Unzip fragments
    13. 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. 14. The End
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