The Molecular Biology Unit at the Instituto de Fisiología Celular provides services to investigators from the Institute and other institutions in the UNAM and outside it. It gives investigators access to a broad spectrum of technologies that are useful in diverse areas of molecular biology. In addition the Unit maintains a small collection of plasmids and bacterial strains, that are provided free of charge. The Molecular Biology Unit is located in laboratory 202 South in the main building of the Institute.

Services:

Oligonucleotide Synthesis

The Molecular Biology Unit synthesise oligonucleotides up to 100 bases, these could include mixed bases for the synthesis of degenerate oligonucleotides. The oligonucleotides are desalted, suspended in distilled water and quantified with a spectrophotometer. The Unit has two PCR-MATE 391 DNA Synthesizer from Applied Biosystems. The synthesis is carried out using beta-cyanoethyl chemistry. Scale of synthesis is 200-nmol. The oligonucleotides synthesised are suitable for direct use in most applications including cloning, hybridization, mutagenesis, PCR, DNA sequencing. The price is 16.50 pesos (approximately 1.15 USD) per base.

Automated DNA Sequencing

The Automated DNA sequencing is carried out on capillary-based electrophoresis sequencers. The Unit utilizes an ABI Prism 310 (1-capillar) and an ABI Prism 3100 (16-capillary) Genetic Analyzers from Applied Biosystems, with Big Dye Terminator Cycle Sequencing chemistry. Samples of single stranded DNA, double stranded DNA, PCR products, cosmids, phage DNA and bacterial genomic DNA, can be sequences with read length up to 700 bases of high-quality sequence data per reaction, providing the template is of high quality. Already labelled and purified sequences are also run. DNA sequencing charges are 200.00 pesos (14.2 USD) per reaction (one template-one primer) for internal users and 250.00 pesos (17.8 USD) for external users, and 70.00 pesos (5 USD) for already labelled reactions.

Cloning

We offer services in DNA cloning and subcloning, PCR products, plasmid construction, and gene fusion. Project will be specifically arranged with researchers for conditions and timing.

Technical support

The Unit provides technical support and training to the academic staff and students of the institute in a variety of Molecular Biology techniques. It also offers diverse courses and talks of particular molecular biology interests.

DNA amplification

The Molecular Biology Unit has three thermal cyclers (thermocyclers) that can be used to amplify DNA segments via the PCR (polymerase chain reactions) process; one of them has a gradient function, which allows to evaluate up to 12 different annealing, elongation, or denaturation temperatures. This is useful when testing suitable temperatures, particularly the annealing temperature for primers, to optimize the PCR reaction. Free of charge.

Real-time PCR technologies

The Molecular Biology Unit has for real-time PCR instrument, ABI Prism 7000 Sequence Detection System from Applied Biosystems. This is a plate-based machine, with 96 well thermal block. This instrument provides investigators with the ability to perform very sensitive, accurate, and reproducible measurements of levels of gene expression. In addition, it can be used for a variety of other applications such as measuring viral load, performing allelic discrimination studies, detecting mRNA splice variants and optimizing PCR conditions. The open format of these instruments allows researchers to work with a variety of fluorogenic systems. The Unit staff can assist in setting up the instrument software to include the cycling profile, sample well assignments. Charge is 120.00 pesos (8.5 USD) per run, for external users.

Radioactivity and Fluorescent Imaging

The Unit has three imagers (STORM 840, Typhoon 8600 and Typhoon 9400) that allow analyzing radioactive and fluorescent samples. The three of them can be used to scan storage radioactive screens that have been exposed to radioactive samples, and two of them (typhoons) can also scan fluorescent and chemiluminescent samples. The system includes data analysis software. The system has very high sensitivity that reduces exposure times to at least half the time required by standard x-ray film. Free of charge.

Imaging Documentation System

An AlphaDigiDoc RT from Alpha Innotech is available for capture and analysis of images. The instrument detects visible and UV light. It can be used for X-Ray films, stained wet gels including protein and DNA gels, as well as for general photography. The system has software that allows image process and optimization, as well as a variety of quantitation analyses. Free of charge.

Flow Cytometry and Cell Sorting

Flow cytometry is a technique which allows determination of cellular characteristics, usually through the utilization of fluorescent markers, and the isolation of cells with unique characteristics based on those parameters. The Molecular Biology Unit has a BD Biosciences FACSCalibur cytometre for user operated data acquisition. This instrument has a 488nm blue laser that allows the analysis of 3 fluorescent parameters to 2 light scatter parameters. It also allows cell sorting. Free of charge.

Pulsed-Field Gel Electrophoresis (PFGE)

Pulse field gel electrophoresis is a technique for resolving chromosome size DNA. By alternating the electric field between spatially distinct pairs of electrodes, separation of DNA molecules up to 2000Kbp in an agarose gel is possible. There is a CHEF-DR II Pulse Field Electrophoresis System from BioRad available at the molecular Biology Unit. Assistance in the equipment set up and the technique is provided. Free of charge.

RNA Interference (RNAi) Technology

RNA interference (RNAi) is a powerful tool for gene function analysis. It allows researchers partially suppress the expression of a specific gene, allowing targeted gene knockdown. Knockdown of target genes with RNAi can be used to elucidate biochemical and cell signalling pathways. The aim of this service is to provide assistance in designing RNAi experiments, from chemistry and material selection to data analysis.

Contact:

• Laura Ongay-Larios: longay@ifc.unam.mx
• B. Sc. Guadalupe Códiz-Huerta: gcodiz@ifc.unam.mx
• M. Sc. D. Minerva Mora-Cabrera: dcabrera@ifc.unam.mx