Arizona Genomics Institute

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
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

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.