Cellular and Molecular Biology
Mounting sections for brain histology
The Anatomy and Histology Facility functions as an extension of the investigators' own labs. Researchers come to use the facility's microtomes; collaborate with facility staff doing animal anesthesia and perfusion; or request tissue sectioning and labeling with stains, antibodies, and DNA/RNA hybridization probes. For each preparation the staff members accomodate the specific needs of the labs they support.
The facility has 1,000 square feet of workspace and is equipped for tissue fixation, sectioning, immuno-labeling, and staining of a variety of mouse brains and other cells and tissues. Facility staff also prepare cultured brain slices and dissociated hippocampal neurons for physiology experiments. There are two chemical fume hoods for fixatives, a cell culture hood, a clean box, and four CO2 incubators for preparing primary cultures. Facility staff have extensive experience with antibody-labeling methods. Some investigators prefer to use the facility's equipment on their own, while others may request staff assistance in performing various histological procedures and methods.
Equipment:
- Cryostats and vibratomes. The facility has two Leica 3050S cryostats, five Leica vibratomes, and a CryoJane tape transfer system. All items are available to investigators for their own use.
- Tissue culture hoods. Culture hoods can be booked by researchers using an online scheduler.
- Microscopes. There is a Zeiss inverted fluorescence microscope for examining primary cultures and an Olympus MVX10, a fluorescence dissecting microscope, for inspecting GFP and antibody labeling in sections.
Services and Training:
- Anesthesia and perfusion-fixation of mice and rats
- Embedding in OTC and agarose
- 5 µm-thick to 50 µm-thick sections prepared on Leica cryostat or vibratome
- Antibody labeling and histochemical staining (Nissl, H&E, special stains)
- Primary cultures of hippocampal neurons
- Preparation of brain slice cultures for physiological recording
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Cell and tissue samples
The Facility provides quality control of equipment, reagents, and cell stocks and perform cell transfection using a variety of reagents and protocols. Through services, training, and collaborations, the Cell and Tissue Culture Facility acts as a stimulating environment for scientific interaction among users and staff, and provides cultures for cell-based assays and tool development.
We provide services in cell culture and cell transfection in a 1,000 square foot facility equipped with nine culture hoods and 15 incubators. The staff is especially experienced in methods of cell transfection. Half of the facility space and equipment is designated for researchers who want to culture and transfect cells on their own. There is also a separate BSL2 room for culturing viruses, such as adeno-associated virus (AAV) and lentivirus.
Equipment:
- Culture hoods and CO2 incubators. Culture hoods can be booked by researchers using an online equipment scheduler.
- Counters, electroporators. The facility has two Beckman-Coulter ViCell cell counters and two Amaxa nucleofectors with a shuttle system for electroporation-based cell transfection.
- Microscopes. A Zeiss inverted AxioObserver fluorescence microscope is available for examining fluorescent protein expression.
Services:
- Amplifying, freezing, and storing cell cultures
- Cell transfection-electroporation with Amaxa Nucleofector
- Cell transfection with lipid reagents, Fugene, Jet-Pei
- Production of AAV and lentivirus
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Services:
- Fast-turnaround DNA sequencing using a 16 channel ABI 3130xl Genetic Analyzer
- DNA isolation services for all scales of plasmid DNA, as well as BAC, YAC, or cosmid
- Rapid small-scale AAV production
- Illumina HiSeq 2000 for next-generation sequencing applications
- Quantitative PCR for genotyping and RNA analysis
- Vector design and construction and development -- either finding the right vector from existing sources or engineering unique vectors specifically for your purposes. The Facility also has the capacity to produce clone collections using these vectors.
- Molecular biology enzyme freezer
- Flexible robotic systems for liquid handling (designed to accommodate processes that become too repetitive in an individual lab)
Concerning sequencing protocols, the Facility can do low-throughput sequencing (and select rush jobs) in-house or can arrange to have high-throughput analyses done through outside vendors.
DNA Sequencing
The Molecular Biology Facility maintains and operates an ABI 3130xl DNA analyzer. This is a 16-capillary machine, capable of processing a maximum of ~384 sequencing reactions over 24 hours of continuous operation. The Facility also offers specialized sequencing services, such as transposon-mediated sequencing and direct sequencing from colonies using rolling circle amplification (RCA). Researchers in need of higher-volume sequencing should contact the Facility manager about outsourcing options.
DNA Preparation & Analysis
The Facility provides a DNA isolation service for purifying plasmid, BAC, YAC, phage and cosmid DNA. Endotoxin-free plasmid DNA preparations are available. Other methods are available upon request. The Facility performs rolling circle amplification (RCA) for rapid enzymatic amplification of DNA that eliminates the need for overnight culturing and minipreps for sequencing applications. The Facility can also provide post-purification services, such as restriction enzyme analysis, sequencing, and purification of inserts for microinjection.
AAV and Lentivirus Production
The Facility offers a service to provide small-scale rapid preparationss of adeno-associated viruses (AAV). The turnaround time for this service is approximately two weeks with a capacity of eight preparations per two-week period. The available serotypes include 1, 2, 5, 7, 8, and ). We provide services for lentivirus production.
Next Generation Sequencing
The Facility offers next generation sequencing (NGS) using an Illumina HiSeq 2000.
Vector Design and Development
The Facility provides a complete cloning and vector construction service for the production of expression, reporter, lentivirus or AAV, or transgene constructs and functional testing. We have experience with utilizing Gateway and recombineering techniques for higher throughput cloning applications.
Lab Automation
The Facility can assist in automating repetitive lab tasks. The facility has two Perkin Elmer liquid handling robotic stations (Janus, Evolution P3) that can be configured for a variety of applications, such as re-arraying clones or functional screens.
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