ELISA Lab

Introduction: (A) Elisa, which stands for Enzyme-Linked Immunosorbent Assay, is a test that can detect diseases in blood and bodily fluids using antigens that bind to the foreign antigens. When antigens trigger an immune system response, millions of antibodies find the antigens and bind to it. The secondary antigens that are enzyme linked are the next ones to bind to the antigens. Next, Chromogenic enzyme substrate is added which will color the wells. If they remain colorless, then they are negative, and if colored, the results are positive. Today, ELISA is used for many different purposes which include pregnancy tests, detecting drug use and disease in people, reassuring that food is labeled correctly and testing indoor air quality. With its rapid test results, ELISA has been very helpful for many diverse purposes. A very important aspect of the ELISA test is the antibodies. They prove to be useful because they can bind really well (with antigens). Some different uses of binding are immunostaining, immunoblotting or western blotting. Different kinds of cells (such as cancer cells) may be identified by the process of immunostaining. A protein's size and quantity may be determined by immunoblotting or western blotting. Antibodies can also be manufactured. Polyclonal antibodies are made by immunizing an animal. An animal (most likely a goat, rabbit, or sheep) would be injected with a purified protein that would allow the animal to make different antibodies to develop within the animal. Serum can then be collected by drawing blood from the animal and removing the blood cells from it. The serum that is isolated from the blood sample of the animal is called antiserum. The final step is to separate the antibodies from the antiserum. These antibodies are called polyclonal. The only down side to polyclonal antibodies is that no two batches will ever be the same. (B) The purpose of the experiment is to find out which two people in the class were originally diseased. This will be done by having everyone share fluids with three other people. After the ELISA test, the results will help to determine who the two disease spreaders were. In reality, the sharing of bodily fluids can be quite common. Someone may cough or sneeze on you with out you knowing and before you know it, you have become a victim to their cold they have shared with you. One in four teenage girls have an STD (from Mr. Chugh's stat of the day) and ELISA would be a way for them to find out. (C) On the first day of the lab, we will obtain a tube of some "bodily fluids". Then, with three other people, we will share our bodily fluids we got and record their tube number and the time at which the sharing occurred. On the second day, we will receive  twelve wells which we will add positive/negative controls, and our shared fluids to. It will then be cleaned out with wash buffer. The next step will be to add the primary antibodies then it will be cleaned out with wash buffer. The secondary antibodies are added next and then also cleaned out again. After it is rinsed with the wash buffer, substrate will be added and show the results. (D) I'm not really able to predict if i will be infected or not because the two diseased people could be anybody. It depends on who I share my fluids with. There are two controls in this experiment: a positive and a negative. The positive wells will be added with fluids that are diseased and should show up as blue. The negative wells will be added with fluids that are diseased and should be clear. For the results in our wells to be sufficient, the positive and negative controls must come out as expected when the substrate is added to it.

Results/Observations: After adding substrate, all twelve of the wells appeared to remain the same. This might mean a source of error occurred while the lab took place. Therefore, it remains a mystery if my lab group and I have been infected.

Discussion: With missing data from table four and five, it is almost impossible to determine who is the original diseased people are. The originally diseased people did share very quickly though because everyone is infected except for a few. The source of error in our lab was washing out the wells before adding the positive/negative controls and our fluids. The primary antigens did not have a chance to bind which made it impossible for the secondary antibody to bind and so on.

Proteomics

Introduction: (a) Genomics is the study of genomes and DNA. Proteomics is the study of proteins. The protein has shown us how the information that has been translated by DNA is used to make an organism. It can be studied to learn how genes that are made of DNA are transcribed by messenger RNA and then transformed into proteins.With the significant help of genomics and proteomics, there have been many discoveries that were once never considered abled to discover or explore. The Human Genome Project sparked the realization of the importance of the necessary variety of proteins. This was what led to a new field of study termed proteomics. Proteomics is more complicated than genomics because the proteome differs from cell to cell and is always changing. Specifically, proteomics studies the functions of proteins, interactions between proteins, their locations in the cell, their expression, and modifications of all proteins within development. The Human Proteome Organization is an international collaboration project which aims to achieve all the above studies of a protein. (b) In this lab, we will will use gel electrophoresis to look at muscle proteins from related fish species. Whether they are closely related or not will be determined at the end of the lab depending on the samples brought in and supplied. We will be able to identify similarities and differences based on the fish's proteins. (c) We will use gel electrophoresis to determine relations of fish.With this, we will need to collect samples from the fish (muscle). Adding buffer (the ingredients needed for the gel to work) to the samples will be the next step before adding the samples into the gel. The gel will run and hopefully the results will turn out to show how closely related each fish is to one another. (d) To insure that the results from gel are accurate, there will be a marker to act as a control for the gel electrophoresis.

GMO:yes or no???

Intoduction: (a) Genetically Modified Organisms or GMO, argued by proponents, are believed to solve the problem of the overuse of pesticides and herbicides on crops that may have long term effects on the environment and human health. GMO's are used to genetically manipulate crop plants to have specific traits. This technique is not new, but what is new is the option to take the genes out of another organism (doesn't have to be from a plant species) for a specific trait and directly place it into crop plants. Some examples include GMO's that are resistant to pests or drought, improved appearance, and increased crop yield. GMO's can be identified by and antibody based test (enayme-linked immucosorbent assay) which can spot the proteins that are made by the GM crops. Another way to detect GMO foods is by a process called the polymerase chain reaction that searches for the specific DNA sequence of a GM food. Today, there is much controversy over GM crops and the absence of the requirement to label GM foods in the US by the government. People who oppose GMO's say that there is the potential for "superweeds" that would occur by the cross pollination of natural weeds and herbicide resistent crops. Also "superbugs"could arise due to the toxins in pest-resistent crops. There is no diversity in the crops (monoculture) which makes them suseptable to a massive wipeout. People could have allergic reactions to certain proteins of the GM crops. Many say there isn't even enough reasearch done to forsee and understand the tinkering of plant diversity and manipulation. (b) The purpose of this lab is to test and see if the food item brought in is genetically modified. Since a label for GM foods is not required in the US, we are unable to identify them. With this experiment, we are able to find out if a favorite food we have been eating is genetically modified or not. (c) DAY 1-The first step will be to extract DNA from food samples. The sample which would be turned into a slurry will then be combined with instagene, the same will be done with the certified non-GMO food product(this will act as a control to test if the lab worked properly). The samples will then be put into a waterbath for five munutes and then refridgerated. DAY 2-Master mix is added to each pcr tube which will be iced. Then DNA template will be added to the pcr tubes but avoiding the pellet from the instagene. The pcr tubes will then be set in a thermal cycler which rapidly heats and cools. This allows the template strands to seperate or denature and the primers to anneal to the denaturated template strands. While in the cycler the DNA polymerase also extends the primers and makes complete copies of each DNA strand. DAY 3-After the pcr tubes have been in the microtube adaptor, loading dye will be added to each sample. The samples will be loaded into the agarose gel. Results are seen after stained in fast blast DNA stain.

CSI:AHS (DNA Profiling)

Intoduction: (a) Restriction enzymes, which come from bacteria, are used in DNA profiling. These enzymes work best under specific buffer and temperature conditions. They act like scissors and cut the DNA according to its recognition of sequences and base pairs. This happens on a restriction site which is what biologists look at to help them identify someones DNA. The DNA that is seperated and observed is done through the process agarose gel electrophoresis. This process organizes the DNA fragments by size and put on and agarose gel slab and then has an electric current pass between the elctrodes. By having this done, the fragments of DNA will be drawn to the positive pole which will allow all the fragments of the same size to form into bands which is seen after the DNA is stained. To compare DNA of different individuals to others, radioactive probes are needed. They recognize and pull together the specific sequences of nucleotides. Evidence for DNA fingerprinting is easily obtained through any material that might contain DNA. Population genetics and genetic statistics are two factors that can possibly aggect the relability of DNA profiling. DNA profiling is used in many other ways besides forensics. Some of the other researches DNA profiling is used for are food identification, the identification of human remains, paternity testing, and the identification of human remains.
(b)The objective of this lab is to identify the criminal by using DNA profiling. We will use the DNA of five suspects to see which of them did the crime. We will be able to observe the DNA fragments and match it up the DNA from the crime scene.
(c)The restriction enzymes will cut the DNA at its palindromes. Then the different lengthed fragments of DNA will be generated which will be the DNA fingerprint. Then we will run a gel and stain the DNA to figure out and observe the DNA and match it up to the crime scene DNA.
(d) In lane 1, the DNA size marker is a control for electrophoresis and will show if this process worked or not.
Results: Once the gel was stained overnight and drained, it showed that lane 5 and lane 2 matched. Lane 2 is the crime scene DNA and lane 5 is suspect 3's DNA.
Discussion: (a) Because of the results, out of the five suspects, katie is the criminal. Her DNA matches the DNA found at the crime scene.
(b)A possible error could have been that when pipeting the different DNA's into the lanes, we could have been careless to put them into the right lane which could lead to the identification of the wrong suspect.

Biofuels

Inroduction:
(a) When biofuels are made, a source that was currently living is turned into reusable energy as fuels. This is different from fossil fuels because the materials used to make them are dead rather than living. The four main types of biofuel development are cellulosic ethanol(cellulose to glucose),ethanol production from sugar and starch sources,syngases(from biomass burning),and biodiesels(from oils). Right now in the fuel industry,the ethanol produced comes from starches that is turned into sugar. There are two problems(a lot of the plant ends up being unused, and is a food source) with this method so researchers are trying to find out how to produce ethanol as fuels by using cellulose instead of using starches. Enzymes lower the activation energy of a chemical reaction which allows it to occur at a much faster rate. Biofuel industries use cellulases(family of enzymes) from cellulose to turn it itno sugars(glucose). It can then be made into ethanol by means of fermentation. The ethanol produced can then be used to power certain engines and combined wtih gasoline to power vehicles.
(b) The purpose of this lab is to identify the reaction rates with and without the prescense of an enzyme. The practical use of this enzyme will prove relevent since the biofuel industry is using enzymes to convert into ethanol.
(c) We will use 5 cuvettes and fill them equally with solution. The chemical reaction will begin when the buffer is added to the controlled solution. The stop solution (enzyme reaction) will kill the cellobiase and turn P-Nitophenal yellow.
(d) I predict that the stop solution will turn the liquid inside the cuvettes more and more yellow. But on the second day of the lab where the mushroom is added, it will be a consistent color of yellow throughout all of the cuvettes.

Results/Obsevations:
On the first day of the lab, when the stop solutions were added to each of the cuvettes, the color of the solution in them became noticably more yellow each time. On the second day with the addition of an enzyme to speed up the chemical reaction, each solution in the cuvettes remained unchanged. All were the same shade of yellow.

Dicussion:
(a) The first day of the lab consisted of different shades of yellow of solution as time went on. This was the progress of an uninfluenced chemical reaction. The second day of the lab consisted of an unchanging shade of yellow solution as time went on. With the addition of an enzyme (mushroom), the chemical reaction occured and finished much quicker.
(b) Some errors for this lab could be that the time sensitive steps were not done properly-did not count the minutes for the addition of stop solution to the cuvettes.

Yogurt Lab Discussion

a) In the first test tube, which was the negative control containing only milk, remained unchanged. In the second test tube, which was the positive control containing milk and yogurt, the texture appeared more like a thick liquid resembling yogurt. It also smelled like yogurt, and so I conclude that the bacteria from the added yogurt affected the milk and turned the whole mixture into yogurt. In the third test tube, which contained the ampicillin and yogurt, the state of the mixture was liquid and smelled like milk. Since the ampicillin is an antibiotic, it must have fought off the bacteria from the yogurt and enabled it to turn into milk. The last test tube contained yogurt and E.coli. This would answer the question as to see if any type of bacteria would be able to produce yogurt. The texture was somewhat resembling to yogurt but the smell was anything but. E.coli is unable to produce yogurt.
b)One possible error that could have occurred could be that the vortex was not properly used and the mixtures in the test tubes were not fully mixed causing complications that would later affect the results.

Yogurt

a) Bacteria-a terrifying species or an essential part of every day life? The answer is both! They are both potentially harmful and a life necessity. Bacteria are everywhere, but are typically too small to be seen by the naked eye. They are prokaryotes which means they are single celled with cell walls. There are many different shapes of bacteria. The three most common shapes are coccus,bacillus, and spirillum. Like all other life forms, bacteria require food(usually sugars). Bacteria uses enzymes to chemically ingest and break down the sugars into their molecules. Bacteria are crucial when it comes to making certain food products like yogurt. Yogurt can be produced when a specific kind of bacterica is added to milk. The mixture then begins to ferment . The bacteria takes in sugars from the milk and releases lactic acid.
b) The objective of this lab is to use a model to utilize Koch's postulates which involves making yogurt. This lab will be a controlled experiment using microbial techniques.
c) There will be four test tubes all containing milk. One of the tubes will have nothing added to it, therefore containing only plain milk. The other tubes will have yogurt, yogurt + ampicillin, and E.coli added to them. After inoculating the milk with the yogurt, we will need to check that the culture is the same.
d) The controls will be tube 1(milk) and tube 2(yogurt). Tube 1 is negative and tube 2 is positive which will verify the results of the lab.