The Wistar Molecular Screening and Protein Expression Facility maintains libraries and equipment for high-throughput screening of small molecule and nucleic acid (e.g. cDNA, siRNA, shRNA) libraries. In addition, the facility is equipped for cell culture and molecular biology. For more information about the facility instrumentation click on a link below:
The facility has the following equipment available:
- Liquid handling robotics to transfer compounds/nucleic acids from library stock plates to assay plates.
- Automated dispensers to rapidly fill assay plates with bulk reagents (e.g. cells, proteins, buffers, etc.).
- A plate reader with absorbance, luminescence, ultraluminesence, fluorescence, fluorescence polarization, time-resolved fluorescence, and alpha screen detection capabilities.
- Automated plate washers.
- A high-content screening reader.
- Surface Plasmon Resonance instrumentation.
- BSL2 Tissue culture capabilities.
Laboratory personnel will provide users with training in the use of the walk-up equipment (e.g. bulk reagent dispensers, plate washers, and plate readers). Users must be trained by facility staff prior to using the instrumentation in an unassisted mode. To schedule a training session, please make an appointment with someone in molecular screening prior to training on the instrumentation in the Facility. All training time on equipment is charged as assisted use.
|Automated pipetting workstations|
|The laboratory is equipped with a Perkin Elmer Janus 96/384 Modular Dispensing Tool (MDT), automated liquid pipetting workstation for compound/reagent transfer and library management. The Janus MDT is equipped with a 384 well nanohead for transferring volumes between 0.05 μl to 0.5 µl, a 384 well head for transferring volumes between 0.5 μl to 25μl with disposable tips, and a 96 well head with capabilities of transferring volumes up to 235μl. The MDT workstation is integrated with a plate stack unit with capacity for up to 50 microplates.The laboratory is also equipped with a Perkin Elmer Janus Verispan 8 channel independent pipetting workstation for use in reformatting libraries (e.g. dilution series, Hit-Picking) and inserting controls on assay plates. The Verispan 8-tip is designed for use with fixed washable tips, disposable tips in 20 μl, 200 μl, and 1 ml sizes, and disposable filter tips in 25 μl and 175 μl sizes, or both.|
|Bulk reagent dispensers|
|A. Biotek microflo|
|The Biotek Microflo uses a peristaltic pump mechanism to dispense liquids in as many as 8 channels, with each nozzle fed by a separate feeding tube. The entire tubing assembly is removable and can be autoclaved when sterility during dispensing is essential. The Microflo can be programmed to fill specific plate columns as desired by the user, and is capable of dispensing 2.0 to 2000 µL in increments of 1 µL. The priming/dead volume for this automated dispenser is about 6-10 mL. In addition the Microflo can be integrated with Biostak plate stacker unit that can accommodate up to 30 plates.|
|B. Biotek µFill|
|This is a syringe-based, automated plate dispenser with a 16-channel manifold with a volume range of 5 μL to 1500 μL for 384-well plates and 10-3000 μL for 96-well plates. The syringe and tubing assembly is removable and can be autoclaved when sterility during dispensing is essential. The priming volume for this liquid dispenser is about 13-20 mL. The μFill can also be integrated with Biostak plate stacker unit that can accommodate up to 30 plates.|
|The ELx405 is an automated plate washer with a 96 channel head that can be used with 96- and 384-well plates. This plate washer also offers a sonicating wash bath for the pins to help minimize clogging and increase the cleaning capabilities. The Elx405 offers the flexibility of integration with the BioStak plate stacker, as necessary.|
|The laboratory is equipped with a PerkinElmer EnVision Xcite Multilabel plate reader with a 40-plate stacking carousel. The Envision is a filter based plate reader capable of measuring standard detection labels in 96, 384, and 1536 well plates, including absorbance, flouresence intensity, luminescence, ultra-luminesence, time-resolved fluorescence, fluorescence polarization (FP), and AlphaScreen (Amplified Luminescence Proximity HomogeneousAssay). A wide selection of filters and dichroic mirrors make most dyes and dye-pairs accessible to the reader. In addition, facility users can use a Perkin Elmer Top Count to read high-density radioactive assays.|
|The Perkin Elmer Operetta is an inverted epifluoresence microscope that utilizes a laser auto-focus option to automate acquisition of high-content images in up to 4 channels (three fluorescent plus brightfield) from 96- and 384-well microtiter plates or slides. It is equipped with a xenon arc lamp excitation source, a motorized objective wheel with bar-coded, interchangeable objectives (2X, 4X, 10X, 20X, 40X), an 8 position excitation filter wheel with matched dichroics, an 8 position emission filter wheel with interchangeable, bar coded filters for maximum detection of fluorescent labels, and a 1.3 megapixel, 14-bit Peltier cooled CCD camera. The Operetta uses Harmony, which includes a seamlessly integrated database for management of images, experimental information, data analysis, and metadata information. This provides the capability for convenient queries using a sortable data tree and cellular image analysis software to select a standard image analysis routine or develop custom analysis sequences to fit a project’s specific needs. Data can be stored locally on a 3.0 TB drive for short periods of time. An additional 10.0 TB network accessible RAID server pre-configured to immediately handle storage, management, and analysis of high-content data using the Columbus web-based interface is available in cooperation with the Bioinformatics Resource and the Center for Systems and Computational Biology. Columbus provides database access to an unlimited number of authorized users, which enables them to use Acapella and develop custom analysis sequences that cannot be achieved using standard analysis algorithms.|
|The Biacore T200 (GE Healthcare) utilizes the Surface Plasmon Resonance (SPR) technology to study biomolecular binding events on a chip surface. This technology has the advantage of being able to work with biomolecules in their native forms, without the need of introduction of label or tag (referred to ‘label-free’). The basic principle involves immobilization of a ligand on a sensor chip followed by continuous delivery of an analyte by a microfluidic system. The SPR-based technology continuously monitors the changes of mass deposition on the detector surface, and allows sensitive and reliable characterization of biomolecular interactions. Any protein, DNA, RNA, lipid, carbohydrate, polysaccharide, cell, virus, drug, drug like molecule (organic or inorganic) can be used as the ligand or analyte. Since the detection system is based on measuring total mass on the sensor chip surface neither ligand nor analyte has to be tagged. The SPR technology can provide quantitative data on:|
The Wistar screening facility has a tissue culture suite approved for work with BSL2 level pathogens. It includes:
- Two 6ft. tissue culture hoods, which can house rapid dispense plate fillers (e.g. μFill) to dispense tissue culture cells into assay plates.
- 4 CO2 regulated tissue culture incubators.
- A benchtop centrifuge fitted with microtiter plate holders and an inverted phase-contrast microscope for use while passaging and preparing cells for assays.
The BSL2 classification of this facility enables utilization of retroviruses (e.g. lentivirus) produced from cDNA and shRNA libraries.
The Facility is equipped for cell culture and molecular biology with:
• Zeiss Axiovert 10 phase-contrast microscope
• 2 class II type laminar flow hoods
• 2 x 25 cu. ft upright incubators (27°C) with multi-position magnetic stirrers designated for insect cell culturing and baculovirus infection of insect cells in spinner flasks
• Floor model ThermoForma refrigerated environmental shaker with capacity for 12 x 2L flasks
• 2 dual-chamber CO2 incubators
• Sorval legend Mach1.6R and Beckman CS-6R low speed bench-top centrifuge
• AKTA purifier FPLC
• Branson 450 sonicator
• 2 x -80°C freezers
The Facility is also well equipped with standard laboratory equipment, including micro-centrifuges, PCR machines, water baths, microwave, refrigirators & freezers, gel electrophoresis units, balances, pH meter, conductivity meter, water purification system and standard supplies for conventional and affinity chromatography approaches to purify recombinant proteins from either bacteria or insect cells.
In addition, the Facility has access to an adjacent common use equipment, including a GE Healthcare Typhoon imager, environmental shakers, Sorvall RC-5B high speed centrifuge and rotors, Beckman XL ultracentrifuge and rotors, GE ImageQuant LAS4000 gel documentation system.