Antibody Testing Introduction

This experiment involves a mock outbreak of an infectious disease within our classroom. After the spreading is over we will use the process of ELISA to see who has the disease. Antibodies are Y shaped proteins that specifically bind to a certain foreign antigen. Millions of antibodies are made and destroyed every ten minutes. There are two types of antibodies primary and secondary. Primary antibodies bind specificially to the antigen, while secondary antibodies are usually made from a different species and bind to the primary antibodies. In the real world Enzyme linked immunosorbent assay is used for pregnancy tests, disease detection in animals and plants, as well as drug use etc.

Procedure:
Antigen is added to a microplate strip and incubates until bound/ washed with detergent.
Next primary antibody solution is added to the wells and allowed to bind/ than the unbound is washed away.
Next the secondary antibodiy is added and allowed to bind to the primary antibody, and than washed away.
Next a chromogenic enzyme is added and allows color to develop if there was binding earlier.

     

Proteanomics Conclusion

Procedure
I was actually absent for this lab but the procedure was pretty similar to most of our other labs. In this case samples of different fish, were cut off placed with a solution of buffer, incubated for a brief period and then heated up to denature the protein. Next gel electrophoresis is run on all of the proteins including, lanes set aside in order to prove or disprove if the first step worked. In our experiment the different lanes of the proteins all showed up except for that of the fly. Unfortunately enough flies have very little muscle tissue and in order to get enough tissue we needed about fifty flies rather than 3 or 4. From our results we were able to determine that the tuna and salmon were most closely related, followed by the shrimp and than the scallop, and than the fake crab, crab, and the fly didnt show up. Some sources of error could be not extracting enough protein, gaining some protein from particles floating in the air, mistaking certain bands because they were soo close together and etc.

Intro to Proteomics

Proteomics is the study of proteins mostly their structures and functions. The comparison of proteins from one species to another supports evolution and helps evolutionary scientists receive a better understanding of how closely a species is related. Proteonomics has risen mostly after the completion of the human genome project that took thirteen years, to be able to see the difference between genes and how they are expressed in certain situations. Recently the study of proteins has expanded to include the study of protein functions, interactions, locations, and modifications. It is hypothesized that the noncoding DNA regulates protein production and expression and because at 3 billion base pairs, we are much more complex than say a worm. The HUPO Human Proteome Organization was created specifically for cataloging all human proteins and function.
The term evolution states that organisms derived from simple single celled organisms and adapted individually to their environment. Mutations that were beneficial were passed on creating different species, that express different phenotypic traits. Muscle proteins consist mostly of myostotin and actin but individual species have different proteins for their specific types of movement.
This lab consists of three steps including 1) Denaturing the protein by heat shock as well as a detergent sodium dodecyl sulfate killing the protein tertiary and quaternary structures. The proteins then are put with a buffer and seperated using gel electrophoresis. Depending on the size of the different proteins from the aquatic animals it shall illustrate how closely our specimens are related. A blue tracking dye is added in order to see how far the proteins went.       

Mitochondrial DNA

In this lab we all had the same bands for this specific gene that we targeted, proving that PCR, and everthing in the lab went right. Possible sources of error could be, the cotton swab in the pipet not filtering enough giving DNA from the air, and some human sources of error could be going to low and taking some of the matrix beads therefore killing the DNA polymerase and stopping the DNA from being amplified.

Intro to Mitochondrial DNA

In Mendelian genetics the offspring of a mother and father should theoretically be 50% mom, 50% father, but in reality this is not true. The mother contributes more in her oocyte with additional organelles and extra DNA in the form of Mitochondria. Mitochondria contains an extra 37 genes and produces the energy the cell needs to function in the form of ATP.
Mitochondria is hypothesized to have originally come from bacteria that was swallowed up by the early cells that eventually became all plants and animals. Mitochondria is interesting because it accumulates mutations approximately 10 times the rate of nuclear DNA. Through this fact scientists have traced human DNA back to Africa 200,000 years ago to a single female. Using mitochondrial DNA is helpful in tracing samples of DNA from a long time ago, or a very weak sample, and can be determined easier by the thousands of the mutations.
In this experiment, PCR is used to amplify a 440 nucleotide sequence within the control region of the mt genome. Cycles are much shorter because a sequence is amplified several thousand times with a nuclear sequence with only two copies. Once again we shall be taking cheek cells obtained from our mouths by chewing gently but vigirously. A salt solution is used once again and then the cells are centrifuged and boiled,. The supernatent DNA solution is combined with buffered solution of Taq polymerase, primers, DNTP building blocksl and MgCl2. It is then placed in a thermal cycler

Intro to Mitochondrial DNA

Results/Observations Genetic Disease

This lab was pretty successful looking around the room I saw that only one group had messed up, and this was due to leaving the electrophoresis machine on too long. The results of my table illustrated that everything was done properly, the control illustrated gel electrophoresis worked, the homozygous controls and heterozygous control illustrated the bands that should be seen and were easily comparable to that of mine, Saba's and Daniel's.  The results about the "disease" included the majority of the class as either carriers or diseased. I was personally lucky to be one of the few that did not have the intron, that in reality does not do a whole lot, at least that we know of. I believe this lab was done effectively and I believe reinvigorates me to hopefully take genetic tests in the future in which I could then appropriately assess my risks, and work towards overcoming/ disproving that we are predestined for anything. I hope others understand that the arguement about nature versus nurture is ridiculous, as in everything their is no black or white, we are who we are because of a complex mixture between genetic determination and the society around us including, exposures to environmental sources.

Disease Gene Lab Intro

Kary Mullis at Cetus Corporation created Polymerase Chain reaction in 1983. PCR is used to create a large amount of DNA in a test tube in order to be able to use it to identify specific genes and spots of interests. Any DNA sample can be used and mass produced such as a drop of blood or hair follicle. DNA  can be used to identify crime victoms, medical diagnosis etc. In this lab we will start on day one by taking DNA from our body such as a cheek swab after rinsing with a salty solution that is than transferred to a test tube. The test tube shall be heated up to over 95 degrees and instagene matrix shall be added so that DNase shall not break up the DNA that we want. This happens for 5 minutes after an incubation period of 10 minutes at 56 degrees celsius.
Next Polymerase Chain Reaction is used to amplify DNA by started with a template, mixing in individual deoxynucleotides, DNA polymerase, Magnesium ions as a catalyst, ogligonucleotide primers, and salt buffers. Copies are made doubling each cycle until there are millions of copies of the same strand of DNA. PCR begins in the denaturation step in which the mixture is heated to 94 degrees celsius for 1 minute seperated the double strand. Next the annealing step has oligonucleotide prmers attach to the complementary strands and ast as primers for polymerae making new complementary strands at 60 degrees celsius. Next the extension step adds the nucleotides to the primer actually making the complementary strand at 72 degrees celsius. These steps form one cycle usually has 40 cycles.
There are 23 pairs of chromosomes in the human genome with about 30k to 50k genes. The other 95 percent is noncoding DNA. In this activity we are lokking for the Alu element in the PV92 intron region. On the gel if both the inserts move at the same speed then they are both either dominant and will show by being slower, or recessive and be the same but moved farther up. If they are one of each then they are a mixture in which in theory if it was a disease the dominate would over ride and they would be a carrier.

GMO Results

In this lab we found that both the orange and the corn samples that we took were genetically modified. This makes sense because the orange that Daniel had was a cutie and corn is almost certainly genetically modified unless otherwise certified within the United States. The controls that were in place thankfully did not need to be used because the lab went splendid. The gel electrophoresis illustrated that the samples with added plant primers went farther and that those with GMO primers all matched up to each other. If they had not shown up in spots two and four we would assume that the samples were not genetically modified but this was not the case. Some sources of error may have been using a pestle and mortar instead of a blender provided us with a lesser amount of sample that then showed up very faintly within the gel. This was not too big of a deal because our results were spot on.

Experimentation Proving Most American Foods are Genetically Modified.

Introduction:

Genetically modified food contains genes implanted from other organisms for a specific purpose. They can be beneficial by providing more nutrition or yield, but the full effects are still unknown. In the US genetically modified foods do not have to be labelled as such which is potentially dangerous for those with allergies, and the public is unable to see the actual pros and cons of the food. Some cons include the potential for creation of superweeds by overuse of cross-pollination with herbicide resistant crops, and the creation of monocultures provides the potential for a single disease to wipe out main food crops such as corn. Also allergies are much more prone by using genes from different species. Some other pros are more storable foods(square), last longer, taste better, be more nutritious, can grow using lesss resources. In this lab we will be testing a variety of foods to determine there upbringing. We could specifically test individually the GMO proteins by useing Enzyme linked immunosorbent assay but unfortunately the genes are different in each organism. Instead we will a Polymerase Chain Reaction to identify the sequences inserted into the GM plant. This test can detect 85 percent of all GM crops because we are not looking for the gene but the plasmid that is transferred from the bacteria to the plant cell.

Procedure:In this lab we will start out by preparing the food and cutting a slice out around all the edges. We will hurt the cell wall and then open up the cell membrane. DNAse will be killed off with instagene maxtrix beads. On day two we wll do the polymerase chain reactions and amplify the DNA. Then the primers, DNA polymerase, nucleotide bases and reaction buffer will be mixed in a single test tube. Then the mixture will be placed in the thermal cycler, where it will cycle from 94 degrees Celsius to 59 degrees Celsius back and forth. The first step is called denaturation which happens at high temperatures. The next is the annealing step in which two template strands will compete with the primers but lose at low temperatures. Lastly the extension step will stay at 72 degrees Celsisus and the DNA polymerase will make copies of the DNA strand. It takes three full cycles that the fragments of precise length are generated. On the third day we will use gel electrophoresis to test with a GMO primer and have a plant primer set as a control.

Playing God; When Do I Learn How to Smite?

In this lab we will perform genetic transformation. Genetically Modified crops are made this way, insulin is created in bacteria for diabetes, and to digest oil spills etc. In this experiment we will be transform bacteria with a gene coding for Green Fluorescent Protein. Therefore the bacteria will glow green under ultraviolet light. In this experiment we will be transferring the gene from one organism to another with the help of a plasmid( contains DNA that bacteria keeps when it needs to adapt quickly.)  The pGLO plasmid contains penicillin resistance as well as the GFP, as well as a gene regulation system to control expression of the protein in transformed cells.
In this lab we will have three main steps to move the plasmid through the ecoli cell membrane
1.Usae a transformation solution of CaCl2.
2. Carry out heat shock.
growing the cells in presence of penicillin
3.Provide the cells with nutrients and an incubation period to allow them to express their new genes.