List of AGI Centers

BAC/EST Resource Center

BAC Library Construction Center

Physical Mapping Center

Genome Finishing and Annotation Center

Sequencing and Research & Development Center

BAC/EST Resource Center

Leader of BAC/EST Resource Center: Dave Kudrna Research Specialist Senior: Wolfgang Golser   Research Technician: Elizabeth Ashley The Arizona Genomics Institute BAC/EST Resource Center provides affordable access to BAC and EST libraries, filters, macroarrays and clones. The BAC/EST Resource Center also serves a primary platform to launch a majority of our BAC library construction, physical mapping, genome and EST sequencing and positional cloning projects. The Center is fully functional and is equipped with a state of the art laboratory and operating 2 Genetix Q-bots (colony picking, library replicating and rearrarying, filter gridding) and 44 -80 freezers (clone archiving). Orders may be placed through our website http://www.genome.arizona.edu/orders/. Recent BAC library projects include: 1) BAC library production and distribution for "Healthy People 2010" (NIH: PI Wing, Co-PIs Tomkins and Soderlund). Recently completed BAC libraries include: wallaby (Macropus eugenii, ME_KBa) and zebra finch (Teaniopygia guttata, TG__Ba). In the pipeline is BAC library construction for nurse shark (Ginglymostoma cirratum) and snail (Biomphalaria glabrata). 2) The Oryza BAC library project: A fundamental public resource to study monocot plant evolution, development and physiology (NSF: PI Wing, PI Co-PIs Soderlund, Tomkins, Jackson & Luo). Current libraries in the BAC construction pipeline are: Oryza rufipogon, O. brachyantha, O. alta, O. officinalis, O. ridleyi, O. punctata O. minuta and O. longistaminata. 3) Collaborative Research: The green plant BAC library project public resources for studying evolution, physiology and development (NSF: PI Mandoli, Co-PIs Wing, Soderlund, Tomkins, DePamphilis & Banks). ). Recently completed: BAC library and small insert genomic library for Volvox carteri (BAC, VCNEBa; small insert, VCNESa). In the pipeline is BAC and small insert libraries for Acorus gramineus and small insert genomic library for Liriodendron tulipifera.

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BAC Library Construction Center

Leader of BAC Library Construction Center: Dr. Raju Jetty Personnel: Dr. Jinke Lin     Dr. Ruifeng He     Dr. Xiang Song     Research Specialists: Jayson D Talag     Nicholas B Sisneros         The AGI BAC Library Construction Center builds high-quality, deep coverage bacterial artificial chromosome libraries for NIH and NSF supported projects and for custom orders. It also builds random-sheared 40 kb genomic DNA fosmid libraries. Libraries are picked, stored and arrayed on high-density filters by the AGI BAC/EST Resource Center. Libraries, BAC and fosmid clones, and filters are distributed and sold through the AGI BAC/EST Resource Center. Apply for BAC libraries through NIH The BAC vector we are using (Available through CUGI) Our BAC library construction protocol

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Physical Mapping Center

Leader of Physical Mapping Center: Dave Kudrna Project Leader for Maize Physical Map Analysis: Dr. Fusheng Wei Senior Research Specialist: Jose Luis Goicoechea The AGI Physical Mapping Center conducts analysis of BAC libraries by means of high throughput BAC DNA fingerprinting using HICF SNaPshot labeling chemistry. The resulting fingerprints can be assembled by the program FPC to create contiguous clone maps which, in turn provide the basis for development of physical DNA maps. Daily capacity HICF SNaPshot samples is 9- 384-well plates per day, using one AB 3730xl DNA analyzer. Fluorescent trace data is analyzed using AB software (Genemapper) and in-house scripts. All fingerprints are assembled into contigs using Fingerprint Contig software (FPC) developed by Dr. Cari Soderlund at AGCoL. Recent research projects include: 10x genome coverage of a representative species from each genome from the Oryza genus for the objectives of the NSF project called "Oryza Map Alignment Project" (www.omap.org). High Information Content physical map of the maize genome in collaboration with J. Messing of Rutgers University (NSF #0211851); 14X map of maize as part of the Maize Mapping Project (MMP, NSF #9872655); An agarose-based physical map of the tomato genome.

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Genome Finishing and Annotation Center

Director of Sequencing : Dr. Rod Wing     Leader of Sequencing Center: Dr. Yeisoo Yu     Lab Manager and Finishing Co-ordinator : Teri Rambo     Finishers : Teri Rambo   Jennifer Currie     Annotation: Dr. Fusheng Wei The DNA Sequencing Center supports project-driven high throughput DNA sequencing of whole genomes, BAC ends, ESTs, and shotgun clones from selected BACs to build genome sequencing frameworks and discover agriculturally important genes. Major projects include the Rice Genome Sequencing Project (Chromosomes 10, 3 and 11), Exploitation of Tomato as a model (Wild potato, cultivated potato, eggplant, petunia, pepper and tomato), Sequence-tagged-connectors projects (Rice, Rice Blast Fungus, Sorghum and Maize) and EST projects (Cotton, Barley, Basil and Rice). The fully equipped Sequencing Center contains two capillary ABIPRISM 3700, 3100 and 3730xl sequencers providing high-throughput production sequencing and finishing environment (~11,520 lanes/week). Finishing Resources on the Internet Finishing Protocol - Wash U. GSC, St. Louis Finishing Manual - Cold Spring Harbor Genome Center Autofinishing Protocol - University of Washington Software manuals - Phred, Phrap, Consed Sequence Analysis Tool Finishing Protocols and Techniques BAC miniprep protocol (Qiagen) PCR reaction and exo-sap clean up Shatter library construction Transposon mediated sequencing TA Cloning of PCR Product Solution list Sequencing Flowchart Finishing at AGI Presentation (Powerpoint Presentation) Poster from PAG XI meeting - January 2003 (Powerpoint Presentation) Chemistries and Reagents BigDye versionII terminators Perkin Elmer Part no. 4314421 dGTP BigDye Terminator kit Perkin Elmer Part no. 4307176 Klentaq DNA polymerase mix Sigma Part no. D6290 TA subcloning kit Invitrogen Part no. 45-0046 Template Generation System Finnzymes (MJ Research) Part no. F700

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Sequencing and Research & Development Center

Leader: Dr. HyeRan Kim     Production Manager: Kristi Collura     Research Specialist: Marina Wissotski Daniel Smart   Jun Pyo Kim     Research Technician: David F Campos     The DNA Template and High Through-Put Technology Center supports project-driven and custom orders for DNA sequencing and small insert genomic library construction. High through-put DNA template production (low copy BACs, or high copy ESTs) is provided by operation of two Quadra96 pipetting robots offering outputs of nearly 10,000 preps per day. Additionally, cycle sequencing reactions are setup and performed using automated pipetting machines and MJ Scientific Tetrad thermocyclers. Sequences are analyzed by AGI Sequencing Center using two ABI 3730 DNA Analyzers. Custom and project small insert genomic (shotgun) libraries are produced by complete or partial restriction enzyme digestion, hydroshear, or sonicator (shatter) into a variety of vectors. AGI Resource Center provides support for robotic picking, gridding and colony handling using Genetix Q-bots.

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