Dr. Raul J. Cano' s research laboratory consists of 5 labspaces comprising 3 separate rooms. The lab is staffed with both Graduate and Undergraduate students working on Master's Thesis Research, detection of Salmonella and Listeria grants, Ancient DNA and Microorganism recovery and analysis, and Microbial Diversity studies. T here are currently 6 "permanent" members, and as always many more students working on more short-term and Senior projects involving microorganisms and Molecular Biology. This tour will introduce to some of the modern facilities we have at our disposal by taking you through how a "typical" day of experiments might go, although around here almost NOTHING is typical!!!

First of all, we might start with a DNA or RNA Extraction. We have multiple areas where this takes place, and here is the "cleanest" of them all. This is the infamous room where we crack amber pieces to recover really old DNA and even some viable microorganisms!!! In the corner can be seen "The Extractor" which is really just an incubator with a rocker for shaking tubes at elevated temperatures. For DNA extractions, our normal protocols include a "quick and dirty" Chelex-100 method, a guanidinium based method adapted from Boom et al, and the tried and true phenol:chloroform method. We perform these extractions on pure cultures, spiked milk, cheese, and cow feces samples, insect tissues(extant and ancient), and the occasional Dinosaur bone and "IceMan" sample(we call him 'Outzi'). Concentrations are calculated from absorbances at 260 nm on a UV:VIS spectrometer. Quality control is assured by exposure of all surfaces to germicidal UltraViolet lights when they are not in use, and by daily washings with 10% Bleach and 70% ethanol.

After finishing our extraction, the next step is usually a PCR, short for Polymerase Chain Reaction. The heart of our PCR facilities is the ultra clean and DNA free "PCR Room" seen here. Except for the DNA on our bodies, no DNA is ever allowed to enter this room. It is kept under constant UV light (except for when in use!!!) and is only used to set up the Master Mix for our reactions. Extra tubes, pipette tips, and solutions are stored here as well to help them remain sterile. We normally run 25-100 uL PCR reactions using a wide range of primers. Some typical regions for us to work with include the IS200 region of Salmonella, the 16S rDNA sequence of many different organisms. PCR reactions are performed using our two Perkin-Elmer thermal cyclers. The 9600 is affectionately called "The Cadillac" because of it's sheer mass yet powerful elegance and the 2400 is known as "Zippy 2" because it's so fast(the 2 is because our first one broke down!!!). Both machines have the advantage of having heated tops making the use of mineral oil obsolete. The thin-walled tubes also assures effective and quick heat transfer from block to tubes. "Hot Start" PCR, where the chilled master mix is added to DNA and immediately added to an 80 degrees Celsius preheated block is routinely used to avoid non specific amplification during the initial ramp to 94 degrees Celsius of the first cycle. We also have a Thermolyne TempTronic Thermocycler and an MJ Research Thermal Cycler(dubbed "Tiny" because it's about 1:20th the size of the 9600!) that are used mainly for RFLP experiments and other short term incubations. The 2400 and 9600 are used for cycle sequencing, RT-PCR, RAPD-PCR, and all of the "regular" PCR reactions.

In order to visualize the reults of our labor, agarose gels are used to electrophoretically separate products. We also have facilities for running both native and SDS-PAGE gels for separation and anlysis of proteins. We typically run 1-2% agarose gels and stain them in a solution of 0.5 ug/mL Ethidium Bromide(DANGER) for 15-20 minutes and then destain them for 10-15 minutes in deionized water. The Ethidium Bromide solution can then be mixed with 1g/L activated carbon and filtered, with the solution now safe enough for sink disposal(This will only work for solutions of less than 0.5 ug/mL ethidium bromide!) The contaminated Carbon can be disposed of as "Hazardous Waste." To view our gels, we use a UVP-ImageStore5000 gel documentation system. It consists of a video camera that captures the UV illuminated gels which give off light in the visible region. From there we can adjust the brightness and contrast to get the best possible picture. The thermal printer allows for permanent copies at around 15 cents a picture, compared to over $1.00 per picture on Polaroid film. We can also save the picture to disk for transfer to computer for further analyis.

After amplification is acheived, we either clone the fragment into a Bacterium to get an "endless" supply of the DNA sequence or we go directly to DNA sequencing. We have ample space for cloning and all other microbiological work, including multiple temperature incubators, an anaerobe chamber, and a full stockroom of media. For sequencing, we use an Applied-BioSystems 373 Automated Sequencer nicknamed "Muhammad" after the technician at ABI that has been a multitude of help in setting it up. This beauty utilizes a laser that reads the dye color in the lane making radioactivity obselete. It also automatically collects the sequence making manual verification unnecessary. Once those sequences are collected, they can be analyzed by one of the 8 computers we have connected to our own little network(5 Macintosh, 2 Sun, 1 PC clone). Our "dinosaur" computer, far from being old and outdated, is named 'Jurassic' and serves as the email server for our lab. With 96 MBytes RAM and 2 GBytes of storage, our Sun SPARC Station10 is more than capable of analyzing and aligning DNA sequences, predicting phylogenetic relationships, and designing primers. Both 'Jurassic' and many of the other computers are directly linked the Internet through Cal Poly's network making literature searches quick and easy.

Well, that's a short tour of our lab and our facilities. Within our Biology Department we also have access to both transmission and scanning electron microscopes, plant and animal tissue culture facilities, and a dedicated darkroom for developing pictures and autoradiographs.