University of Florida | ICBR

Services at a Glance

454, BIA, BLAST, BlastQuest, Custom Sequencing, Genotyping, Illumina, myMAP, myCAP, Protein Sequencing, SNP Genotyping,…

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Bioinformatics Services (Li Liu) Related Pages

High Throughput Bioinformatics Tools

Large scale batch BLAST search: Equipped with Paracel BlastMachine running on a computer cluster with 28 processors, we are able to perform BLAST search against standard NR, NT, SwissProt databases and custom databases.
EST assembly: EST sequences will be assembled using Paracel Transcript Assembler, which goes through quality clean, vector masking, repeat masking, contamination sequence removing, clustering and assembling processes.
Bacterial genome annotation: For complete or draft bacterial genome, FGENESB Bacterial Genome Annotation Package is able to predict genes for tRNA, rRNA and mRNA, operons and COG functions.
Mascot Protein Identification: Running on a server with 8 processors, mass-spectrometry data will be searched against standard IPI, NR, NT databases and custom databases.

Microarray Analysis

QA/QC
Normalization
Statistical analysis - Parametric, non-parametric
Functional analysis - GeneOntology, pathway
Gene Marker Identification -feature selection, classification and prediction

Custom Databases and Web Applications

BlastQuest: A web-based database application that allows users to store, manage and analyze EST sequences.
Genome Project Web Site: A domain we reserved for genomic, transcriptomic and proteomic projects conducted at UF. We design, develop and maintain the web pages for your projects.
Collaborative Annotation Portal - myCAP: myCAP provides an environment for descriptive genomics. It features controlled access to large-scale annotation projects that allows browsing, searching, editing and version tracking of annotation data and comparative genomics data.
Microarray Annotation Portal - myMAP: Under development

Custom Microarray Design

Prepare target sequences- EST assemblies, gene predictions.
Annotate target sequences
Identify non-redundant gene sets from the target sequences
Probe design
Array layout design

Bioinformatics Consulting

First meeting is free
Sequence Analysis - Database search, sequence alignment, phylogenic analysis, annotations, etc.
Microarray Analysis - Image analysis, normalization, statistical analysis, functional analysis, etc.
System Administration - Software installation on UFGI-STATS server

Bioinformatics Laptop Rental

Currently installed software - GeneMapper 4.0, PathwayStudio
Proteomics - Under development
Metagenomics - Under development
Comparative Genomics - Under development
Comparative Transcriptomics - Under development

Cellomics Services

Electron Microscopy & Bioimaging (Byung-Ho Kang) Related Pages

Scanning and transmission electron microscopy, providing investigators with consultation, training, access to instrumentation, and fee based services.

Preparation and examination of biological samples for transmission and field emission scanning electron microscopy. This includes:

Conventional and microwave sample preparation
Pre/post-embedding cytochemical and immunocytochemical localizations
Whole mount preparations
Critical point drying
Vacuum evaporation and sputter coating.

Flow Cytometry (Neal Benson,MS) Related Pages

User Access to Flow Cytometry Instrumentation Analysis of User-Prepared Samples by Staff Sorting (separation) of Sub-populations of Cells Graphical and Statistical Analysis of Flow Cytometry Data Archiving of Flow Cytometry Data Files Presentation Graphics from Flow Cytometry Data Training in Instrument Operation Consultation in Assay Design: A consultation is required before initiating any new projects. For more information, contact Neal Benson, MS

Hybridoma (Linda Green) Related Pages

Development of Monoclonal Antibodies

Immunization

2-3 female, Balb/CByj mice, 6-8 weeks old are injected with antigen, often spanning a concentration range of 20 - 100 ug per mouse (determined in advance for each antigen). Adjuvants (MPL + TDM emulsion-RIBI or Titermax Gold) are used to enhance the immune response. Additional immunizations are given and test bleeds from immunized animals are obtained according to customized protocols.
Immune serum is tested for specific antibody responses using ELISA, Western Blot or some other method such as immunohistochemistry, immunoprecipitation or bioassays.
Immunizations and testing continue until an adequate response is attained. When the mice have a high titer and are secreting IgG, and the assay conditions have been optimized, a fusion is scheduled.
Four days prior to the fusion, the selected mouse is injected one final time, usually without adjuvant. Mice not selected for fusions are kept in reserve. The reserve mice may be bled out for polyclonal serum . It is also possible to harvest and process the spleens, freezing the cells for possible fusions in the future.

Fusion

The selected mouse is anesthetized, exsanguinated and euthanized. Spleen cells are fused with a non-secreting mouse myeloma cells line at a prescribed ratio using 50% PEG 1500 (Polyethylene glycol) as the fusing agent. Fused cells are seeded into 96 well plates in HAT selective media. After several days, hybridoma colonies should be visible. The cells are maintained by feeding with fresh media as needed. Once the cells near confluence, an aliquot of supernatant is removed from each well and tested (usually by ELISA) for the presence of antigen-specific antibody. Cells that test positive are expanded for additional application-specific screening. Cell lines that have the desired antibody specificity will be frozen. Investigators typically select at least1-2 of these cell lines for cloning.

Cloning:

Uncloned hybridoma cells lines in the log phase of growth are seeded at a single cell per well density in enriched media over a feeder layer of irradiated mouse 3T3 fibroblasts. The feeder layer forms a matrix which allows the cells to form visible intact colonies after approximately 5 days in culture. The wells of the cloning plate are scored for single colony growth. The supernatants from single colony wells are assayed to determine whether the desired antibody is present. After further rounds of screening, at least two cloned cell lines are expanded for deposit into our cell bank. One of these two cell lines is designated as the primary clone and the other as the secondary. Mycoplasma testing and isotype determination will be performed on the primary clone.

Production of Monoclonal Antibodies

For small to medium amounts of monoclonal antibody (10 - 15milligrams) the recommended method is to use a special partitioned culture vessel (Integra CELLine flask) and media formulated to maximize monoclonal antibody production. This media is supplemented with a low IgG-containing fetal bovine serum. Cells grow to a very high density in the cell compartment allowing fairly high concentrations of antibody to accumulate. Supernatant is harvested from the cell compartment once a week for three weeks. If a larger amount of antibody is needed, this system can remain in operation for a longer time. The concentration of monoclonal antibody in this harvested material is typically in the range of 0.3 - 1.0 mg/ml. If smaller amounts of antibody are required, a protocol using standard culture flasks of various sizes is available.

Purification of Monoclonal Antibodies

Purified antibodies are quantified by absorbance readings at 280 nM, evaluated by SDS-PAGE and provided to the customer in the buffer of their choice.

Protein G purification of mouse IgG containing cell culture supernatant.
Purification of mouse IgM containing cell culture supernatant
Purification of Ig from polyclonal sera
Purification of other subclasses available upon request

Recombinant antibodies

UF investigators may contract with this laboratory for generation of human scFv reagents using the Griffin 1 or Tomlinson I & J libraries that have been licensed from the developers.

Additional services:

Deposit

Cell lines from outside UF or those developed by UF investigators may be deposited in our cell bank. Mycoplasma testing is required

Antibody labeling

Biotin
Other labels: please inquire.

Immunoassay and antigen design assistance

Genomics Services

454 Sequencing (Regina Shaw) Related Pages

Services Include:: Library construction and titration

Sample preparation for titration for samples that need no library construction

Production sequencing using small (40x75 mm) or large (70x75 mm) PicoTiterPlates; The service includes de novo assembly or mapping of 454 sequence data to a reference genome. Consultation regarding 454 sequencing project design is also available.

Gene Expression (Gigi Ostrow, PhD) Related Pages

Research Services:

Total support for custom microarray development beginning with DNA sequence analysis, ORF finding, and sequence annotation, through microarray fabrication using Agilent's "SurePrint" technology.
Agilent Microarray scanner for imaging Agilent's lineup of preprinted or custom design microarrays, or any 2-color microaaray manufactured in the industry-standard 1x3 format.
Facilities for complete processing of the most recently available high-density Affymetrix GeneChips.

Experimental Services:

Experimental design and consultation
Target hybridization, scanning and both initial and advanced data analyses with or without cDNA and cRNA syntheses using the Affymetrix GeneChip system
DNA and RNA analysis using the Agilent 2100 BioAnalyzer
Imaging of preprinted or custom design microarrays or any 2-dye microaaray compatible with any 1x3 glass slide format

Genetic Analysis (Ginger Clark, MS) Related Pages

Wildlife Forensics: The ICBR Genetics Analysis Lab has developed a series of forensic tests using molecular techniques designed to assist wildlife enforcement officers in combating poaching and other illegal activities involving wildlife. The lab maintains, and continually adds to, an extensive database on sea turtles, whitetail deer, turkeys and alligators, as well as some fresh water turtles such as alligator snappers. We can identify species, gender, and the genetic profiles of most species of interest to the state.
Genotyping: Genotyping, or fragment analysis, is one of the most valuable tools for developing genetic profiles. Virtually any fragment labeled with a fluorescent molecule can be analyzed for size variations on automated sequencers. The ICBR Genetic Analysis Laboratory (GAL) at the University of Florida accepts fragment analysis samples, such as STRs, T-RFLPs, SNups, AFLPs, etc., from on campus users as well as off campus users. Samples submitted for analysis will be run on either a 96 capillary MegaBACE (Amersham) or an ABI 3730 Automated Sequencer. The raw data are analyzed with GeneMapper (ABI) or GeneMarker (Soft Genetics) software. You receive the data as either a raw file to load into your analysis program, or as a PDF file of sized, labeled chromatograms. Go to the How Do I? page for instructions on sample submission.

Realtime PCR (Li Zhang, PhD) Related Pages

: Our facilities are equipped with state-of-the-art instrumentation and our experienced staff provides a valuable resource to help our customers, from both academic and commercial laboratories, to meet their research goals and objectives.; The Core Facility seeks to provide the highest quality Quantitative PCR services in a timely, convenient, and cost-effective manner to support the research community. These services are available to investigators at the University of Florida, other academic institutions, and commercial entities. During the last few years there has been a substantial increase in use of this shared resource reflecting the quality, convenience and value of the service provided.; We support both SybrGreen and Taqman reaction chemistries. With Taqman chemistry, a labeled probe is included in the reaction to enhance the specificity and sensitivity of target sequence detection. With SyberGreen chemistry, amplified product is detected by its interaction with the SyberGreen fluorescent dye, which selectively binds to double-stranded DNA templates. SyberGreen chemistry is less expensive, but generally requires more effort to optimize reactions. Although the Taqman chemistry is more expensive, it generally provides better specificity without the need for extensive optimization. We also offer primer and probe design services.; We offer qPCR for gene expression or SNP analysis. Researchers may choose to have the Core handle the entire project from reverse transcription to finished results, or use our existing qPCR instruments such as, 7700, 7900HT or 7500 for running plates and have the core do data analysis for you. We also offer training in experiment design and data analysis for qPCR users. Consultation is also available for every service we offer, which includes primer design, sample preparation, data interpretation and analysis, and problem solving.; We perform high-quality, cost-effective, core services for molecular and cell biology research. We apply years of experience, the highest quality reagents, and state-of-the-art instrumentation to your research. We work hard to make your research more successful. We don't charge more than it costs us to run each service.

Sanger Sequencing (Savita Shanker, PhD) Related Pages

Custom Sequencing

Automated DNA sequencing using the Applied Biosystems Model 3130 Genetic analyzer is a one-lane sequencing process that uses four-dye fluorescent labeling methods and a real-time scanning detector. These sequencers automatically perform the electrophoresis, call the bases and archives the data. Using single- or double-stranded DNA (plasmids), or PCR-generated DNA fragments, prepared according to our suggested guidelines, the experienced technical staff of DSEQ will:

run the cycle sequencing reactions,
load them on the gel
run the electrophoresis,
and analyze the sequence

using ABI 3130 DNA sequencers. Depending on the purity of DNA preparation, and the length of the capillary used, anywhere from 900-1100 nucleotide bases will be provided at the end of a specified turn-around time, usually 2 to 3 working days depending on the number of reactions requested. The researchers receive the sequence data and electropherograms through the dNAtools web interface. Server address is http://langsat.biotech.ufl.edu. A hard copy of the waveforms showing base assignments and advice and information regarding base-calling techniques and sequence alignments, are also available. Sequence data can be sent by fax, e-mail or provided on user-supplied diskettes. A full sequencing service, which includes sequencing of an entire DNA fragment (2 Kb or greater) by primer walking is also available. Those interested should contact Savita Shanker about estimates of cost and time of completing the sequencing project.

96 well and 384 well Sequencing:

For large sequencing needs, samples can be provided as glycerol stock in 96 well format, colonies on petri dishes or 96well PCR amplicons.

96 and 384well template preparation using RCA protocol

Bacterial cell growth
Denaturation reaction
Overnight amplification with RCA mix

96 and 384well PCR amplicon purification using exo-sap clean up protocol DNA Sequencing in 96well format

Big dye-terminator sequencing reactions
Reaction clean-up prior to loading
Electrophoresis using ABI-3730-XL

DNA sequencing in 384-well format

ET terminator sequencing reactions
Reaction clean up prior to loading
Electrophoresis using Amersham's MegaBACE.

The researchers will receive all sequence data and chromatograms via the Internet, through our Geospiza Finch web interface. Please contact Savita Shanker, PhD for further details.

SNP Genotyping (David Moraga, PhD) Related Pages

Current Services include:

Whole genome SNP genotyping (from 10⁵-10⁶ tag SNPs per chip)
Copy number/Loss of heterozygosity genotyping
Focused content SNP genotyping
- Standard panels (cancer SNP, DNA test, non-synonymous human, MHC)
- Human and mouse linkage analysis panels
- GoldenGate custom panels
- iSelect custom panels
Gene expression analysis in a multiplex format

Please visit the Illumina website under the "products and services" link for more details.

Coming soon!

DNA methylation profiling
DASL assay for expression studies on paraffin-embedded samples

Proteomics Services

Researchers are strongly encouraged to discuss strategy with us before proceeding with sample preparation.

2-D Difference Gel Electrophoresis (Marjorie Chow, MS) Related Pages

Services include:

1D and 2D SDS PAGE
Native gel electrophoresis
2D DIGE (Cy2, Cy3, and Cy5 fluorescent dye labeling)
2D gel-based PTM analysis using modification-specific fluorescent stains
Gel image analysis with Progenesis SameSpot, PG220 and DeCyder v6.5 software
Robotic platform for gel spot excision, digestion, and MALDI plate spotting
Electroblotting to PVDF membranes
Isoelectric focusing
Gel imaging system for fluorescent stain (Typhoon)

Peptide Synthesis (Alfred Chung, MS) Related Pages

Services Include

Custom synthesis 2-50 amino acids
Cyclic peptides
Peptide labeling with fluorescent tags
Chromogenic substrate synthesis
Combinatorial peptide libraries
Phosphopeptides
Purification of synthetic peptides
Quality control of synthetic peptides by HPLC & mass spectrometry

Mass Spectrometry and Protein Characterization (Scott McClung) Related Pages

Hybrid quadrupole-TOF (ABI QSTAR XL), hybrid quadrupole-linear ion trap (ABI 4000 QTRAP), ion trap (Thermo LCQ Deca) and MALDI-TOF/TOF (ABI 4700 Proteomics Analyzer)

These instruments are available for the identification of proteins from gel or solution
Available for complex protein mixture analysis using 2D-LC and tandem mass spectrometry
Available for the characterization of posttranslational modifications
Available for differential protein expression profiling via stable isotope labeling techniques
Available for De novo peptide sequencing

MALDI-TOF/TOF (ABI 4700 Proteomics Analyzer)

This instrument is available for peptide or protein molecular weight determination.
Training is available for self-service use

Database Search Engine and Quantitative Proteomic Software

Mascot 2.1
Scaffold 1.6
ProQuant 1.1

Protein Characterization (Scott McClung) Related Pages

Protein Sequencing

N-terminal Edman degradation
Internal fragment sequencing
In-gel & on-PVDF membrane digestion of proteins
Microbore & capillary HPLC separation of fragments

Amino Acid Analysis

Pre-column derivatization analysis
Protein quantification

Biomolecules Interaction Analysis (BIA) (Marjorie Chow, MS) Related Pages

Label free interaction analysis in real time (Biacore)

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