MOLECULAR BIOLOGY SERVICES AND SHARED INSTRUMENTATION

Module directors

James Hurley, PhD
+1 206 543 2871
jbhhh@uw.edu

Maureen Neitz, PhD
+1 206 543 7998
mneitz@uw.edu


Module research scientists

Jing Huang, PhD
+1 206 543 5959
jinghu@uw.edu

Jonathan Linton, PhD
+1 206 685 4961
jl07559@uw.edu


Lab technician

Gail Stanton
+1 206 685 4961
gails3@uw.edu


RESOURCES AND SERVICES

Shared instrumentation in ophthalmology (contact: Maureen Neitz)

Real Time PCR Machine
GloMax Luminometer
FC2 Alpha Innotech Gel Documentation System

 
Shared instrumentation in biochemistry (contact: James Hurley)

BioRad PCR Machine
S1000 BioRad PCR Maching
Gel Doc XR
Zeiss epifluorescent stereomicroscope
Axiocam MRM digital fluorescence imaging system

 
Genotyping (contact: James Hurley)

The scientists and equipment associated with the genotyping service are located within Dr. Hurley's lab (HSB J631D), which is conveniently located near the animal facilities where the majority of NEI funded investigators house animals.  This makes drop-off of animal tissues for genotyping convenient and efficient.  Services include:
       
• Performing routine isolation of genomic DNA from mouse tails or zebrafish fins.

• Performing genotyping protocols already established by individual investigators.

• Developing new genotyping protocols including primer design, reaction optimization, and troubleshooting.

• Routinely performing genotyping reactions, assay products using gel electrophoresis, document results, and provide investigators with a report, typically within 24 hours of receiving DNA or tissue samples.

• Providing training on and access to the Zeiss epifluorescent stereomicroscope, Axiocam MRm digital fluorescence imaging system, and standard imaging station for "molecular phenotyping" of transgenic zebrafish carrying genes encoding fluorescent proteins.

 
Monoclonal antibody production (contact: Maureen Neitz)

Investigators will provide the antigen for immunization and screening. Dr. Huang will immunize mice, verify a polyclonal antibody response, fuse mouse myeloma cells with cells from the spleen of the immunized mouse, screen hybridoma cells via ELISA, immunoblot analysis, or immunocytochemistry.  She will then clone hybridoma cells from soft agar or by limiting dilution, type the antibody, create and maintain hybridoma cell lines using tissue culture techniques to obtain monoclonal antibodies, and wean hybridoma cell lines from serum-containing media to serum-free media.  Dr. Huang will determine the type of immunoglobulin (e.g., IgM or IgG, and the subtype), and will examine the specificity of the antibody via immunoblot analysis, ELISA, immunocytochemistry or immunoprecipitation. 
 
Localization of mRNAs via in situ hybridization (contact: Maureen Neitz)

Using investigator-supplied cDNA or other suitable vectors for probe production, Dr. Huang will prepare probes, prepare tissue sections or blocks, incubate probes with tissues, process for observation of the results, and prepare images for publications or slides.   She will also train personnel in labs of participating investigators in this procedure upon request.
 
Single and/or double label immunocytochemistry (contact: Maureen Neitz)

Dr. Huang will fix the tissue and cut cryosections or prepared whole mounts, probe with one or two antibodies and/or DAPI, and prepare images for publications and/or slides.   She will also train lab personnel of participating investigators in this procedure upon request.
 
Apoptosis assay (contact: Maureen Neitz)

Cells undergoing apoptosis are detected with the TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) assay.  Apoptosis assays are performed by Dr. Huang using ApopTag®(Serologicals Corporation) or antibodies to Caspases 3 or 8.  She will train personnel in labs participating in this procedure upon request.
 
Proteomics (contact: Maureen Neitz)

This service utilizes the Mass Spectrometry Center, located in the Department of Medicinal Chemistry at the University of Washington.  The center operates as a cost center and is currently overseen by David Goodlett, PhD. The facility is staffed by four scientists and is equipped with eleven mass spectrometers ranging from MALDI-TOF instruments to a Fourier transform ion cyclotron instrument.  The Mass Spectrometry Center staff will assist with designing and performing experiments, and with the interpretation of data.  The facility will also assign fee-for-service mass spectrometer analyses according to investigator need.  The service is designed to accommodate the needs of investigators with episodic needs for protein sequence analysis, identification, or characterization not covered in their research grants.