Cell Culture and Genotyping

The Cell Culture and Genotyping Service (CCGen) aims to facilitate the implementation of state-of-the-art advances in cell culture, genotyping and gene expression technology, by providing at i3S researchers selected services, expert consultation and training in cell culture, genotyping and gene expression techniques. In CCGen Cell Culture Lab we have available tailor-made conditions for a variety of cell lines, primary cells and virus culture, including lentivirus-based transfection service. The provided training is mandatory. We collaborate with interested groups in the implementation and optimization of mouse genotyping protocols includes DNA extraction, DNA fragment amplification, detection, analysis and interpretation of results. Through the Animal Facility the researcher accesses our on-line ordering of mouse genotyping service request (about 100 mouse genotyping protocols in routine). We also offer an urgent genotyping results for all investigators that require to do a select primary culture or who needs a specific time point. We have available for Gene Expression service 3 qPCR equipment’s with thermal validation and quality control. We also support the analysis of RNA integrity by automated electrophoresis and collaboration in the all interpretation data.

Applications

Gene Expression

The qPCR was develop to determine rapidly and accurately the relative amount of RNA or DNA in a sample. To accomplish rapid quantitation and efficient PCR conditions, reactions were design to produce small PCR products (often under 80-150 bp) using short cycles (usually under 30 seconds for a complete cycle). Data is generate as the reaction is running (threshold cycles, CT) or immediately after cycling (melt curves and standard curves generated from CT values). No post-reaction gel is required. Real-time PCR or quantitative PCR (qPCR) is routinely used in laboratories to help identify pathogens, genotype infectious agents, and for cancer diagnostics.

The Experion is an automated platform for nucleic acid and protein analysis, which incorporates LabChip technology into an integrated system that performs multiple electrophoresis steps in one. The automated gel electrophoresis system is used for analysis (quantity and quality) of RNA, DNA and protein, reduces exposure to hazardous formaldehyde and ethidium bromide, decreases the amount of sample needed and provides a reliable system for documentation of results. This instrument serves as a complement to the qPCR, allowing for the fast and economic assessment of RNA quality.

Genotyping

The service does the implementation and optimization of animal (mouse, zebrafish…) genotyping protocols, including DNA extraction, DNA fragment amplification, detection, analysis and interpretation of results (about 100 mouse genotyping protocols in routine).

Cell Culture

Offers expert consultation and training in cell culture with establishment of tailor-made conditions for culture of a variety of cell lines and primary cells, maintenance of hygienic conditions of the cell culture lab and mandatory training of all new users.
We provide Zoe Fluorescent Cell imager, an inverted imaging system, with light field and three fluorescent channels suitable for image cell culture in routine applications.

Resources

Gene Expression

  • iCycler iQ5 Real-Time PCR (Bio-Rad) in operation since 2008;
  • CFX Real time System (Bio-Rad) in operation since 2014;
  • Experion, Automated Electrophoresis System (Bio-Rad) in operation since 2010.

Genotyping

  • TProfessional Basic Gradient 96 thermocycler (Biometra) in operation since 2008;
  • Termomixer compacto (Eppendorf) in operation since 2008;
  • Termomixer compacto (Eppendorf) in operation since 2011;
  • Dry Bath FB15103 (Fisher Scientific, Lda) in operation since 2014;
  • Centrifugue 5415R (Eppendorf) in operation since 2008;
  • Centrifugue MULTIFUGE X1R 230V RESEARCH (Thermo Scientific) in operation since 2011;
  • Gel Tray, 15x20 cm and 10x15 cm (BioRad) in operation since 2008;
  • GelDoc EZ System (BioRad) in operation since 2012;
  • Maxwell 16 DNA/RNA/Protein Automated Extractor (Promega) in operation since 2007.

Cell Culture

  • Six CO2, heated incubators (Heraeus, Hera Cell), distributed by three independent rooms in operation since 2004;
  • Three laminar flow hoods (Heraeus, Hera Safe), one per each room in operation since 2004;
  • An inverted microscope (Zeiss, Axiovert 25) in operation since 2003;
  • One heated water bath (DIG 26L AMB +5 TO 99C. SA, Grant) in operation since 2016;
  • One refrigerated centrifuge (Heraeus, Megafuge 1.0R) in operation since 2004;
  • ZOE Fluorescent Cell Imager (BioRad) in operation since 2015. Minispin plus (Eppendorf) in operation since 2007.