Quantitative Molecular Biology (QMB)

The QMB Core will facilitate the quantification of mRNA and DNA requirements for faculty and staff. This core also offers access and consultation to technologies designed to characterize and quantify changes in gene expression. The QMB core is located at the MSC and adjacent to the University of Wisconsin Biotechnology Center which offers sequencing and various proteomics services.

Services

Primer Design

Investigators unfamiliar with PCR primer design can consult with the QMB CORE to obtain the primers best suited for PCR needs, including specialized primers for nested PCRs and primer design with restriction sites for sub-cloning. Primer design strategies to amplify homologous sequences from species where there may not be a published target sequence are utilized. Design criteria are now established to utilize MIQE guidelines.

Quantitative real time PCR (qPCR)

RT-qPCR is performed using the Quant Flex Studio 7 (QS7) RT PCR machines using Taqman SYBR Green I (as the primary dye) on 96-well standard, 96-well fast, 384-well standard, and 384 well microfluidic card platforms (microfluidic cards are designed by the user and custom synthesized by Applied Biosystems/ThermoFisher).  Data generated from each run is exported into an Excel Spreadsheet

Allelic discrimination of mouse genomic DNAs

Screening of mouse genomic DNAs is conducted using single nucleotide polymorphism (SNP) TaqMan genotyping technology using the Quant Flex Studio 7 machine with preformulated or custom primer sets from ABI.

Quantification of promoter transcription activity by reporter gene analysis

The QMBC offers instrumentation to monitor Luciferase reporter gene activity for transient transfection assays or for assays from transgenic mice carrying appropriate reporter genes.  Assays are read in a Biotek Neo2 multiplate reader that is capable of reading firefly and Renilla Luciferase for the Duo-Glo gene reporter system from Promega.  Users are expected to provide plates, tubes and reagents for use in the machines.

Protein Quantification

Protein levels are measured by gel-electrophoresis and immunoblotting.  Target proteins are identified using Infra-red tagged secondary antibodies and quantified using the LiCor Odyssey CLx infrared imaging system. Quantification is normalized to total protein levels by using the LiCor REVERT total protein staining kit, unless requested otherwise.

New QMB Equipment

Zeiss PALM Microbeam Laser Capture Microdissection (LCM) system laser microdissection and analysis for DNA, RNA and protein isolation from live cells and slides.  This instrument enables the precise isolation of cells for micro-genomic, -transcriptomic and -proteomic analyses.

Luminex MAGPIX multiplex analyzer performs up to 50 different tests for a single sample volume.  Kits are available for immunology, immune response, metabolism/endocrinology, toxicity, for various species.

Please contact Hongyu Noel to receive training on the LCM and the Magpix.

Link to Equipment

For questions regarding Quantitative Molecular Biology services, please contact:

QMB Core Director

Robert W. Nickells, Ph.D.
571A Medical Sciences
1300 University Ave.
Madison, WI 53706
Phone: 608-265-6037
Email: nickells@wisc.edu