University of Puerto RicoRISE ProgramCayey, Puerto RicoBIOL 4997 – 2013 Laboratory SummariesImportance and Pipetting PracticeThe micropipette is a laboratory instrument used to transfer a measuredvolume of a liquid. It is used in different analytical techniques. There are twotypes of micropipettes that are generally used: air-displacement pipettes andpositive-displacement pipettes. We used air-displacement micropipettes that arepiston-driven and dispense an adjustable volume of liquid from a disposable tip.During this workshop we learned how to correctly use micropipettes that werefrom different volumes with their correspondent tips. By using differentvolumes we also had to decide, according to the amount of liquid that wasrequired, which micropipette was the most adequate to have a precise, or asclose as possible, sample. We were also tested on our ability to followinstructions and to work in teams. To work in a laboratory doing research,means to become part of a web of knowledge where everybody, as coworkers,shares it to achieve a common goal. That is why micropipeting is a fundamentaland basic technique for biology and general research, as well as collaborativework.Microscopy and PhotomicrographyWhen things are not visible to the naked eye we use microscopes. Thefirst microscope was made by Hans and Zacharias Janssen in 1590 and fromthere many other scientists like Robert Hooke and Anton Van Leeuwenhoek haveused them to make some of the most important discoveries for humanity.Microscopes can be separated into several classes, mostly because of what theyuse to generate an image. The three most well known are optical microscopes,electron microscopes (scanning and transmission), and scanning probemicroscopes. During this workshop we used optical microscopes with differenttypes of light microscopy and micro-techniques. I was assigned to observeTillandsia trichomes under dark field microscopy using a whole-mount micro-
technique. Dark field microscopy uses light from an angle where it onlyilluminates the specimen and its background is completely dark. Microscopedevelopment has abled humans to evolve and also to increase and expand ourknowledge, not being retained by size anymore. Without microscopes, humanitywouldn’t be the same; as for they allow us to see a complete different world thatis not visible to the naked eye.Workshop University of North Carolina (UNC) - From DNA to ProteinThe principal purpose of this workshop was for us to learn and applytechniques in molecular biology using previous biological knowledge andcombining it with hands-on-work. We refreshed our knowledge of the centraldogma, from DNA to proteins and expanded it with specific terminologies thatlead us to understand more clearly the different mechanisms of our body. On thefirst day, we extracted our own DNA utilizing products that are available to thegeneral public like Gatorade and Contact Lens cleaning solution. During thesecond day we did PCR to determine if we had samples from patients who wereinfected with diabetes. Viewing the PCR results requires you to make an agarosegel that revealed that if our patient was positive or not for diabetes. The thirdday, we run an SDS-PAGE gel to determine if some samples presented aLysosomal Storage Disorder (LSD). After denaturing the protein and running thegels, in the PCR machine that used an electricity current to separate de particles,we found out that our patient was positive for LSD, specifically diabetes. Thesetechniques enable us determine clearly different diseases that may affect ourDNA, and also to identify our DNA. To round up the workshop we were orientedabout graduate school, different roles that exist in a lab, and the research ourmentors were doing. From DNA to protein was a multidisciplinary workshopthat pushed us to be better scientists.Nanotechnology and Electron MicroscopyDr. Otaño’s workshop was about nanotechnology and electronmicroscopy. During this experiment we created a polymer nanotube utilizing theelectrosipining process. To form the nanotube a polymer solution was dispensedfrom a needle with a capillary tip at a high voltage. Then as the fiber formed it
was deposit on the fiber mat over a counter electrode. This fiber is thenprocessed on an air and a vacuum or gas atmosphere at 600 oC. The processedfiber was then taken to a machine where the process of sputtering covered itwith Gold atoms. In this machine, the Gold plaque is bombarded with Argon andthe ion made the gold atoms spread through the chamber, which are collectedover the substrate. This substrate was then examined in a SEM, which provided a3D visualization of the nanotubes. With this experiment bioactive fibers can bedeveloped to treat bones in different medical situations and also MEMS (Micro-Electro-Mechanical devices) can be developed.Column Chromatography and SDS-PageDr. Bansal’s workshop objective was to isolate Plasminogen Activators(PA) from a mammalian cell culture broth using magnetic affinitynanoabsorbents. These magnetic nanoparticles (MNPs) are mostly used becausewhen magnetic separation is combined with affinity binding it condenses thepretreatment and the column chromatography stages into one single isolation.During the pretreatment of the particles the weighed MNPs were combined withPABA binding buffer and then were sonicated to equilibrate the particles at aspecific pH. Then the supernatant was decanted magnetically using magnets.During the separation process the pretreated MNPs were added to the HeLa cellculture broth and then they were separated magnetically and washed threetimes. After the washes, the protein was separated from the particles to obtainan elute. All of the samples, from each step, were taken to a machine to read theirabsorbance at 280 nm (nanometers) and days (how many?) later an SDS-Pagewas performed. Protein isolation is performed to obtain purer proteins that canbe use for dietary supplements, structure studies, as therapeutics or as catalysts.Protein – Protein Interactions: an Insilico approachDr. Maldonado’s workshop was about Insilico drug discovery anddevelopment. This process is based on four primary steps. During the first part, aprotein was downloaded from a 3D structure database and a grid was created toselect the part from which a benzene map was developed. From this map,
optimal targets were selected and during the second part, after an interactionanalysis using the “Ligand Scout” program, a pharmacophore model wasgenerated. During the third part, a primary screening of the pharmacophoremodel was run and at the end one compound was selected. These results wereconverted from .sdfto .mol2. After uploading the .mol2 file to the “Cyberduck”program this file was again converted but now to a .pdbqt file. Now, in the fourthpart, this file is run in “AutoDockvina” where a docking/screening is performedand then analyzed to determine how the model should be refined. This process ishelpful because it gives science a more precise alternative and also a more costeffective way to determine the correct compounds needed to create new drugs.