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Genotyping and Real-Time PCR

Director:  Joseph R. Testa, PhD, FACMG
Manager: Emmanuelle Nicolas, PhD
Emmanuelle.Nicolas@fccc.edu
215-728-2489
P3013
Location & Phone: P3013
215-728-2489 (manager);
215-214-1696 (staff)
Pricing & Scheduling: Contact Emmanuelle Nicolas for pricing and scheduling.

 

Function

Genotyping and Real-Time PCR performs PCR-based services including genotyping for investigators who maintain colonies of transgenic and knock-out mice in their research, mutations, and epigenetic alterations analyses and quantification of nucleic acids. New assays are developed based on investigators' needs. All primers and probes for in-house developed assays are kept in a database ready for sharing by all users.

Description

The Genotyping & Real-Time PCR Unit provides platforms and expertise for targeted DNA and RNA analyses (including quality control, SNP analysis, quantitation of gene expression, quantitative analysis of alternative spliced transcripts, validation of the effectiveness of siRNA transcription silencing, CpG methylation analysis, preparation of samples for Sanger sequencing by Genewiz, and HLA genotyping). It houses a bank of ~1,500 assays as a shared resource. This service also provides cost-effective mouse genotyping (from DNA extraction from mouse tails and toe clips) to support the Laboratory Animal Facility (LAF) and multiple investigators who maintain colonies of transgenic and knockout mice. The data are entered into an on-line mouse genotyping database which tracks animal inventory for each strain of mice. New assays are developed based on the changing needs of the investigators. All assays can be full service (from assay design to data analysis). The sharing of assays has facilitated consultations on RT-PCR issues such as guidelines for publication or normalization of data.

Genotyping and Real-Time PCR occupies approximately 600 square feet on the second floor of the Cancer Prevention Pavilion. Laboratory equipment includes the PSQ96MA (Pyrosequencing), CEQ8800 (fragment analysis), an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System, two ABI7900 (Real-Time PCR), a Corbett Robotics PCR pipetting robot, an Agilent Bioanalyzer (lab-on-a-chip), a Nanodrop spectrophotometer, an Eppendorf MasterCycler Gradient Thermal Cycler, an MJ Research PTC-225 Tetrad Thermal Cycler and a BioRad Dyad Thermal Cycler. A Baker SterilGuard II hood is dedicated exclusively to the preparation of PCR reactions. An Eppendorf 5075VAC System is used for automated DNA extractions from tissues and cells. General laboratory equipment includes horizontal electrophoresis apparatus for DNA analysis, a Corning pH meter (model 340), refrigerator and freezers (-20°C and -80°C) for reagent and specimen storage, non-circulating water bath and dry block incubators. Equipment rooms are located across the hall and provide access to a UV box, alpha imaging system for gel documentation, and a Baker SterilGuard II hood dedicated to biohazard sample handling.

Real-time PCR assay validation. Each reverse transcriptase real-time PCR experiment includes a 5-point, 4-fold dilution standard curve with a calibrator sample to ensure the linearity of the reverse transcription step and to measure the PCR efficiency.
This facility suppports Fox Chase Cancer Center's Cancer Center Support Grant (CCSG) from the National Cancer Institute.

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