University of California, Berkeley View Institution's Website 45 articles published in JoVE Biochemistry Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation Trinity Cookis1, Paul Sauer2,3, Simon Poepsel4,5, Bong-Gyoon Han6, Dominik A. Herbst1,2,6, Robert Glaeser6, Eva Nogales1,2,3,6 1Department of Molecular and Cell Biology, University of California, Berkeley, 2California Institute for Quantitative Biology (QB3), University of California, Berkeley, 3Howard Hughes Medical Institute, University of California, Berkeley, 4Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital, University of Cologne, 5Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 6Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy. Biology Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors Brian Benyamini1, Meagan N. Esbin1,2, Oscar Whitney1, Nike Walther1, Anna C. Maurer1 1Department of Molecular and Cell Biology, University of California, Berkeley, 2Biophysics Graduate Group, University of California, Berkeley Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction. Neuroscience Bringing the Clinic Home: An At-Home Multi-Modal Data Collection Ecosystem to Support Adaptive Deep Brain Stimulation Gabrielle Strandquist1, Tomasz Frączek2, Tanner Dixon3, Shravanan Ravi3, Raphael Bechtold4, Daryl Lawrence5, Alicia Zeng6, Jack Gallant7, Simon Little3, Jeffrey Herron8 1Computer Science and Engineering, University of Washington, 2Neuroscience, University of Washington, 3Neurology, University of California, San Francisco, 4Bioengineering, University of Washington, 5Bioengineering, University of California, Berkeley, 6Biophysics, University of California, Berkeley, 7Psychology, University of California, Berkeley, 8Neurological Surgery, University of Washington The protocol shows a prototype of the at-home multi-modal data collection platform that supports research optimizing adaptive deep brain stimulation (aDBS) for people with neurological movement disorders. We also present key findings from deploying the platform for over a year to the home of an individual with Parkinson's disease. Biology Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes So Goto1,2,3, Michael Frost4, Christine Wildsoet1 1Herbert Wertheim School Optometry and Vision Science, University of California, Berkeley, 2Department of Ophthalmology, Osaka University Graduate School of Medicine, 3Department of Ophthalmology, Tokyo Medical Center, National Hospital Organization, 4Department of Optometry and Vision Science, School of Optometry, University of Alabama at Birmingham We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses. Environment Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India Karthikeyan D. Rajamani1, Sankar Sambandam1, Krishnendu Mukhopadhyay1, Naveen Puttaswamy1, Gurusamy Thangavel1, Durairaj Natesan1, Rengaraj Ramasamy1, Saritha Sendhil1, Amudha Natarajan1, Vigneswari Aravindalochan1, Ajay Pillarisetti2, Michael Johnson3, Joshua Rosenthal*4, Kyle Steenland5, Ricardo Piedhrahita3, Jennifer Peel6, Maggie L. Clark6, Dana Boyd Barr5, Sarah Rajkumar6, Bonnie Young6, Shirin Jabbarzadeh7, Ghislaine Rosa8, Miles Kirby9, Lindsay J. Underhill10, Anaite Diaz-Artiga11, Amy Lovvorn5, William Checkley12, Thomas Clasen5, Kalpana Balakrishnan1 1Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), 2Division of Environmental Health Sciences, University of California, Berkeley, 3Berkeley Air Monitoring Group, 4Division of International Epidemiology and Population Studies, National Institutes of Health, 5Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 6Department of Environmental and Radiological Health Sciences, Colorado State University, 7Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, 8Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 9Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, 10Cardiovascular Division, Washington University School of Medicine, Washington University, 11Centro de Estudios en Salud, Universidad del Valle de Guatemala, 12Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes. Biology Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans Kenneth Trang1, Rahul Bodkhe1, Michael Shapira1 1Department of Integrative Biology, University of California, Berkeley Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition. Biology Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii Marco Valerio Rossi-Stacconi1, Xingeng Wang2, Amanda Stout2, Lorenzo Fellin1,3, Kent M. Daane4, Antonio Biondi5, Judith M. Stahl4, Matthew L. Buffington6, Gianfranco Anfora1,3, Kim A. Hoelmer2 1Research and Innovation Centre, Fondazione Edmund Mach, 2Beneficial Insects Introduction Research Unit, Agricultural Research Service, United States Department of Agriculture, 3Center for Agriculture, Food and Environment, University of Trento, 4Department of Environmental Science, Policy and Management, University of California Berkeley, 5Department of Agriculture, Food and Environment, University of Catania, 6Systematic Entomology Laboratory, Agricultural Research Service, United States Department of Agriculture Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid. Biochemistry Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers Mowei Zhou1, Shadan H. Abdali1, David Dilworth2, Lifeng Liu2, Benjamin Cole2, Neha Malhan1, Amir H. Ahkami1, Tanya E. Winkler1, Joy Hollingsworth3, Julie Sievert3, Jeff Dahlberg3, Robert Hutmacher4,5, Mary Madera6, Judith A. Owiti6, Kim K. Hixson1, Peggy G. Lemaux6, Christer Jansson1, Ljiljana Paša-Tolić1 1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 2DOE-Joint Genome Institute, Lawrence Berkeley Laboratory, 3Kearney Agricultural Research and Extension Center, University of California Agriculture and Natural Resources, 4West Side Research and Extension Center, University of California, 5Department of Plant Sciences, University of California, Davis, 6Department of Plant and Microbial Biology, University of California, Berkeley The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops. Bioengineering Simple, Affordable, and Modular Patterning of Cells using DNA Katelyn A. Cabral1, David M. Patterson2, Olivia J. Scheideler1, Russell Cole3, Adam R. Abate4,5,6, David V. Schaffer7,8, Lydia L. Sohn9, Zev J. Gartner2,6,10 1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Department of Pharmaceutical Chemistry, University of California San Francisco, 3Scribe Biosciences, 4Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 5California Institute for Quantitative Biosciences, University of California San Francisco, 6Chan Zuckerberg Biohub, University of California San Francisco, 7Department of Chemical & Biomolecular Engineering, University of California Berkeley, 8Helen Wills Neuroscience Institute, University of California Berkeley, 9Department of Mechanical Engineering, University of California Berkeley, 10Center for Cellular Construction, University of California San Francisco Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion. Biology Measurements of Physiological Stress Responses in C. Elegans Raz Bar-Ziv*1, Ashley E. Frakes*1, Ryo Higuchi-Sanabria*1, Theodore Bolas1, Phillip A. Frankino1, Holly K. Gildea1, Melissa G. Metcalf1, Andrew Dillin1 1Department of Molecular and Cell Biology, University of California, Berkeley Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress. Bioengineering High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay Jana A. Hassan1, Ilea J. Chau-Ly1, Jennifer D. Lewis1,2 1Department of Plant and Microbial Biology, University of California, Berkeley, 2Plant Gene Expression Center, United States Department of Agriculture The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance. Bioengineering Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics Jonathon M. Muncie1,2, Roberto Falcón-Banchs1, Johnathon N. Lakins2, Lydia L. Sohn1,3, Valerie M. Weaver2,4,5,6 1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Center for Bioengineering and Tissue Regeneration, Department of Surgery, University of California San Francisco, 3Department of Mechanical Engineering, University of California Berkeley, 4Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 5UCSF Comprehensive Cancer Center, Helen Diller Family Cancer Research Center, University of California San Francisco, 6Department of Anatomy, Department of Bioengineering and Therapeutic Sciences, and Department of Radiation Oncology, University of California San Francisco Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification. Genetics Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis Carly V. Weiss1,2, Julie N. Chuong3, Rachel B. Brem1,3 1Department of Plant and Microbial Biology, University of California Berkeley, 2Department of Biology, Stanford University, 3Buck Institute for Research on Aging Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait. Genetics Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis Jingxun Chen1, David McSwiggen2, Elçin Ünal1 1Department of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, 2Department of Molecular and Cell Biology, Li Ka Shing Center, University of California, Berkeley This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis. Genetics Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms Behnom Farboud*1,2, Erin Jarvis*1, Theodore L. Roth*3,4,5,6, Jiyung Shin*1,3, Jacob E. Corn1,3, Alexander Marson3,5,6,7,8,9, Barbara J. Meyer1,2, Nipam H. Patel1,10, Megan L. Hochstrasser3 1Department of Molecular Cell Biology, University of California, Berkeley, 2Howard Hughes Medical Institute, University of California, Berkeley, 3Innovative Genomics Institute, University of California, Berkeley, 4Biomedical Sciences Graduate Program, University of California, San Francisco, 5Department of Microbiology and Immunology, University of California, San Francisco, 6Diabetes Center, University of California, San Francisco, 7Chan Zuckerberg Biohub, 8Department of Medicine, University of California, San Francisco, 9UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 10Department of Integrative Biology, University of California, Berkeley Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms. Chemistry Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching Matthew R. Hauwiller1, Justin C. Ondry1, A. Paul Alivisatos1,2,3,4 1Department of Chemistry, University of California-Berkeley, 2Department of Material Science and Engineering, University of California-Berkeley, 3Kavli Energy NanoScience Institute, 4Materials Sciences Division, Lawrence Berkeley National Laboratory Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations. Genetics Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere Tuesday Simmons1, Daniel F. Caddell1, Siwen Deng1, Devin Coleman-Derr1,2 1Department of Plant and Microbial Biology, University of California, Berkeley, 2Plant Gene Expression Center, USDA ARS Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species. Environment Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions Jian Gao1,2, Joelle Sasse1,2, Kyle M. Lewald1,2, Kateryna Zhalnina1,2, Lloyd T. Cornmesser1,2, Todd A. Duncombe3, Yasuo Yoshikuni2, John P. Vogel2, Mary K. Firestone4, Trent R. Northen1,2 1Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 2Joint Genome Institute, Department of Energy, 3Joint BioEnergy Institute, 4Department of Environmental Science Policy and Management, University of California This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions. Chemistry Low Pressure Vapor-assisted Solution Process for Tunable Band Gap Pinhole-free Methylammonium Lead Halide Perovskite Films Carolin M. Sutter-Fella*1,2,3, Yanbo Li*1,4, Nicola Cefarin1,5,6, Aya Buckley1,7, Quynh Phuong Ngo8,9, Ali Javey2,3, Ian D. Sharp1, Francesca M. Toma1 1Joint Center for Artificial Photosynthesis, Chemical Sciences Division, Lawrence Berkeley National Laboratory, 2Electrical Engineering and Computer Sciences, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, 5Department of Physics, Graduate School of Nanotechnology, University of Trieste, 6TASC Laboratory, IOM-CNR - Istituto Officina dei Materiali, 7Department of Chemistry, University of California, Berkeley, 8Materials Science and Engineering, University of California, Berkeley, 9Joint Center for Artificial Photosynthesis, Lawrence Berkeley National Laboratory Here, we present a protocol for the synthesis of CH3NH3I and CH3NH3Br precursors and the subsequent formation of pinhole-free, continuous CH3NH3PbI3-xBrx thin films for the application in high efficiency solar cells and other optoelectronic devices. Developmental Biology A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents Megan M. Johnson1, Julianna Deardorff1, Kimberly Parra1, Abbey Alkon2, Brenda Eskenazi1, Elizabeth Shirtcliff3 1Center for Environmental Research and Children's Health (CERCH), Berkley School of Public Health, University of California, Berkeley, 2San Francisco (UCSF) School of Nursing, University of California, San Francisco, 3Human Development and Family Studies, Iowa State University Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth. Genetics Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing Joanna M. Feehan*1,2, Katherine E. Scheibel*1, Salim Bourras3, William Underwood4, Beat Keller3, Shauna C. Somerville1 1Department of Plant and Microbial Biology, University of California Berkeley, 2John Innes Centre, Norwich Research Park, 3Department of Plant and Microbial Biology, University of Zürich, 4USDA-ARS Sunflower and Plant Biology Research Unit Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing. Bioengineering Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes Jackson T. Del Bonis-O’Donnell1, Abraham Beyene1, Linda Chio1, Gözde Demirer1, Darwin Yang1, Markita P. Landry1,2 1Department of Chemical and Biomolecular Engineering, University of California Berkeley, 2California Institute for Quantitative Biosciences (QB3), University of California Berkeley We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements. Chemistry Combining Solid-state and Solution-based Techniques: Synthesis and Reactivity of Chalcogenidoplumbates(II or IV) Günther Thiele1, Carsten Donsbach2, Isabell Nußbruch2, Stefanie Dehnen2 1Department of Chemistry, University of California, Berkeley, 2Fachbereich Chemie, Philipps-Universität Marburg and Wissenschaftliches Zentrum für Materialwissenschaften The synthesis of chalcogenidoplumbates(II,IV) via the in situ reduction of nominal "PbCh2" (Ch = Chalcogen) and via a solid-state reaction and subsequent solvothermal reactions is presented. Additionally, reactivities of plumbate(II) solutions are portrayed, which yield the heaviest-known CO homolog known to date: the µ-PbSe ligand. Biology Visualizing Stromule Frequency with Fluorescence Microscopy Jacob O. Brunkard1, Anne M. Runkel2, Patricia Zambryski2 1Plant Gene Expression Center, Agricultural Research Service, USDA, 2Department of Plant and Microbial Biology, University of California, Berkeley Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described. Chemistry Synthesis of a Water-soluble Metal–Organic Complex Array Purnandhu Bose1, Pradip K. Sukul1, Omar M. Yaghi2,3,4, Kentaro Tashiro1 1International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 2Department of Chemistry, University of California–Berkeley, 3Materials Sciences Division, Lawrence Berkeley National Laboratory, 4Kavli Energy NanoSciences Institute at Berkeley, University of California–Berkeley A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented. Developmental Biology Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks Priscilla A. Erickson1, Nicholas A. Ellis1, Craig T. Miller1 1Department of Molecular and Cell Biology, University of California, Berkeley Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback. Biology Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton Nicholas A. Ellis1, Craig T. Miller1 1Department of Molecular and Cell Biology, University of California, Berkeley The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species. Developmental Biology Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation John D. Blair1, Helen S. Bateup1, Dirk F. Hockemeyer1 1Department of Molecular and Cell Biology, University of California, Berkeley Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance. Immunology and Infection Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells Meghan Zuck1,2, Caroline Feng2, Kevin Hybiske1 1Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, 2Program in Infectious Diseases, School of Public Health, University of California at Berkeley A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics. Engineering Failure Analysis of Batteries Using Synchrotron-based Hard X-ray Microtomography Katherine J. Harry1,2, Dilworth Y. Parkinson3, Nitash P. Balsara2,4,5 1Department of Materials Science and Engineering, University of California Berkeley, 2Materials Science Division, Lawrence Berkeley National Laboratory, 3Advanced Light Source Division, Lawrence Berkeley National Laboratory, 4Department of Chemical and Biomolecular Engineering, University of California Berkeley, 5Environmental Energy Technology Division, Lawrence Berkeley National Laboratory Synchrotron-based hard X-ray microtomography is used to image the electrochemical growth of dendrites from a lithium metal electrode through a solid polymer electrolyte membrane. Engineering Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities Han Sae Jung1,2, Hsin-Zon Tsai1, Dillon Wong1, Chad Germany1, Salman Kahn1, Youngkyou Kim1,3, Andrew S. Aikawa1, Dhruv K. Desai1, Griffin F. Rodgers1, Aaron J. Bradley1, Jairo Velasco Jr.1, Kenji Watanabe4, Takashi Taniguchi4, Feng Wang1,5,6, Alex Zettl1,5,6, Michael F. Crommie1,5,6 1Department of Physics, University of California at Berkeley, 2Department of Chemistry, University of California at Berkeley, 3Department of Chemical and Biomolecular Engineering, University of California at Berkeley, 4National Institute for Materials Science (Japan), 5Materials Sciences Division, Lawrence Berkeley National Laboratory, 6Kavli Energy NanoSciences Institute, University of California at Berkeley and Lawrence Berkeley National Laboratory This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential. Bioengineering Production and Targeting of Monovalent Quantum Dots Daeha Seo*1,2,3, Justin Farlow*4,5,6, Kade Southard1,4,7, Young-wook Jun1,7, Zev J. Gartner4,5,6,7 1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications. Immunology and Infection Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen Kwame Twumasi-Boateng1, Maureen Berg1, Michael Shapira1 1Department of Integrative Biology, University of California, Berkeley The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage: sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses. Bioengineering Lipid Bilayer Vesicle Generation Using Microfluidic Jetting Christopher W. Coyne1, Karan Patel1, Johanna Heureaux1, Jeanne Stachowiak3, Daniel A. Fletcher4,5, Allen P. Liu1,2 1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired. Chemistry Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals Karthish Manthiram*1,4, Brandon J. Beberwyck*2,4, Dmitri V. Talapin5,6, A. Paul Alivisatos2,3,4 1Department of Chemical Engineering, UC Berkeley, 2Department of Materials Science and Engineering, UC Berkeley, 3Department of Chemistry, UC Berkeley, 4Materials Sciences Division, Lawrence Berkeley National Laboratory, 5Department of Chemistry, University of Chicago, 6Center for Nanoscale Materials, Argonne National Laboratory A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented. Engineering Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting Corsin Battaglia1,2, Karin Söderström1, Jordi Escarré1, Franz-Josef Haug1, Matthieu Despeisse1, Christophe Ballif1 1Institute of Microengineering (IMT), Photovoltaics and Thin Film Electronics Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes. Biology Fabrication and Use of MicroEnvironment microArrays (MEArrays) Chun-Han Lin1,2, Jonathan K. Lee1, Mark A. LaBarge1 1Life Science Division, Lawrence Berkeley National Laboratory, 2Department of Comparative Biochemistry, University of California, Berkeley A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis. Neuroscience Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat Elizabeth D. Kirby1, Kelly Jensen2, Ki A. Goosens3, Daniela Kaufer1,4 1Helen Wills Neuroscience Institute, University of California Berkeley, 2Office of Laboratory Animal Care, University of California Berkeley, 3McGovern Institute for Brain Research & The Department of Brain and Cognitive Science, Massachusetts Institute of Technology, 4Integrative Biology Department, University of California Berkeley Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain. Biology Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay Zhiying Ji1, Luoping Zhang1 1Genes and Environment Laboratory, University of California, Berkeley A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described. Biology Label-free in situ Imaging of Lignification in Plant Cell Walls Martin Schmidt1, Pradeep Perera1, Adam M. Schwartzberg2, Paul D. Adams3, P. James Schuck2 1Energy Biosciences Institute, University of California, Berkeley, 2Molecular Foundry, Lawrence Berkeley National Laboratory, 3Physical Biosciences Division, Lawrence Berkeley National Laboratory A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species. Biology OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides Markus Günl1, Sascha Gille1, Markus Pauly1,2 1Energy Biosciences Institute, University of California, Berkeley, 2Department of Plant and Microbial Biology, University of California, Berkeley A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself. Biology Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila Iris C. Howlett1, Mark A. Tanouye1,2 1Department of Molecular and Cell Biology, University of California, Berkeley, 2Department of Environmental Science, Policy Management, University of California, Berkeley Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system. Biology In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy Ena Ladi1, Paul Herzmark1, Ellen Robey1 1Department of Molecular and Cell Biology, University of California, Berkeley We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy. Biology Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain Kyle Kurpinski1, Song Li1 1Department of Bioengineering, University of California, Berkeley It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates. Biology Ex vivo Mechanical Loading of Tendon Krishna Asundi1, David Rempel2 1Department of Bioengineering, University of California, Berkeley, 2Department of Bioengineering, University of California, Berkeley; Division of Occupational Medicine, University of California, San Francisco A new in vitro system for simultaneously loading four tendons in culture is described.
