Catch My Disease: Disease Gene Lab

Introduction
a. Background- In this lab, we will be testing DNA to see if an individual carries a gene for a disease. In order to do so, one must test the genome, which is our hereditary code. We will be extracting our own DNA and mass producing in through PCR (Polymerase Chain Reaction). The necessary components for PCR are primers, nucleotides, DNA polymerase, and the existing DNA template to be replicated. DNA is beneficial because it can replicate large amounts of DNA that were formerly not able to be replicated. After replicating the gene, we will run the DNA through a gel, which will show us if we carry the gene for the disease.
b. Purpose- This procedure is important because it could be used to detect if one carries the genes for a disease before symptoms actually occur. This could mean lives and large amounts of money being saved by early detection of diseases.
c. Summary- On Day 1 of the lab, we will be extracting DNA. first we will collect DNA from our cheek cells and break open the cell membranes. We will also add instagene matrix to kill DNAse, which could harm the DNA. On Day 2, we will be using PCR to replicate the small amount of DNA that we extracted. Primers used in PCR target the "disease gene" in the genome. On Day 3, we use gel electrophoresis to diagram the results of the test.
d. Hypothesis- I hypothesize that one of us will have the "disease gene".

Procedure
On day 1, we rinsed our mouths vigorously with saline for 30 seconds. We transfered 1 ml of the saline rinse into a micro test tube containing 200 microliters of InstaGene matrix. We then centrifuged the tubes for 2 minutes, and saw a pellet of cell appear at the bottom of the tube. After pelleting the cells, we poured off the saline and re suspended the pellet by vortexing the tube. Next we micropipetted 20 microliters of of cells into the tube containing InstaGene, and then again vortexed the tube. Then we incubated the tube at 56 degrees Celsius for ten minutes, but at 5 minutes we vortexed the tubes before returning them back to the bath. After, we placed the tubes in a boiling water bath for 5 minutes. Then, we vortexed the contents for 5 minutes and refrigerated the tubes overnight. On day 2, we centrifuged the tubes for 2 minutes. Then we transferred 20 microliters of the DNA template into the PCR tube being sure to not transfer any matrix beads. We also added 20 microliters of the master mix into the PCR tube. Finally we placed the tubes into the thermal cycler. On day 3, we obtained our PCR tubes and centrifuged them for 3 seconds. Then we added 10 microliters of loading dye into the PCR tube and mixed it gently. After we pipeted 10 microliters of the marker into lane 1 of the gel, homozygous (+/+) control in lane 2, homozygous (-/-) control in lane 3, and heterozygous (+/-) in lane 4. In lanes 5, 6, 7, and 8, we each placed 20 microliters of our DNA. After we ran the gel at 200 volts for 3 minutes. When the gel was complete, it was stained and then we analyzed it the next day.

Results

Discussion
After analyzing our results, it turns out that I do not have the "disease" that we were testing for. Although there were no obvious errors in our lab, one source of error could be pipetting DNA as well as matrix beads into the PCR tube and then having the DNA be destroyed. 

Is My Strawberry a Frankenfood?: GMO Foods Lab

Introduction
a) Background- GMO's are genetically modified organisms, and can be used in various scientific settings, often in agriculture, such as in the development of making genetically modified foods, that could be resistant to disease or frost or having a higher amount of vitamins in it. GMO's are made by taking a plasmid with the gene of interest (a tumor inducing plasmid) and putting it into agro bacterium. Then we will place the agro bacterium in a plant cell. The plasmic will take the DNA and will transcribe it, creating a genetically modified plant. GMO's are identified throught two techniques, ELISA and PCR. We are using PCR in  this lab, where we identify sequences of DNA that have been inserted into the GM plant. DNA fragments can be isolated from highly processed foods. GMO's are controversial because some people identify genetically modified organisms as "unnatural" and have the potential to create organisms that will be resistant to normal toxins. Many also worry about potential allergic reactions to GMO's and think that organic foods are better for the environment.
b) Purpose- The purpose of this lab is to take an organism and determine if it is genetically modified or not by comparing it to a known organic organism. The technique of PCR is used regularly in the scientific world today, for example in forensics and genetic studies.
c) Procedure- First we will mortar and pestal the cell of an organism to break down its cell wall. Then we will place the crushed cell in a 99 degree Celcius water bath to break down the nuclear and cell membranes. we will also have to add Instagene Matrix beads that kill DNAse enzymes, otherwise the DNAse will come into contact with the DNA because the cell wall is broken and will destroy it. Next, we will use the PCR process using two primers, one to target plant DNA (the control) and one to target GM DNA. Finally, we will use gel electrophoresis to visualize the gene of interest and determine the presence/absence of the desired PCR products.
d) Hypothesis- In this lab we are testing if the organism we bring in is genetically modified or not.
Procedure
On Day 1, we will label two tubes with Test Food 1 and Test Food 2. After we weighed out about 0.9 grams of non-GMO food (corn flour) and put it in the mortar, along with 10 milliliters of distilled water. We ground up this mixture for around two minutes and then followed by adding 5 milliliters of distilled water. Next we pipetted 50 microliters of the slurry into the tube containing InstaGene. After, we prepared our tube for our second test food, strawberries, using the same procedure. Then we flicked both tubes and placed the tubes in a 95 degree Celsius water bath for 5 minutes. We followed by placing the tubes in a centrifuge for 5 minutes and then we refrigerated the tubes over night. On Day 2, we labeled six PCR tubes. Tubes 1 and 2 contained 20 microliters of non-GMO food control DNA. Tubes 3 and 4 contained 20 microliters of test food DNA. Tubes 5 and 6 contained 20 microliters of GMO positive control DNA. After we labeled these tubes, we placed them in the foam float on ice. Then we added 20 microliters of plant master mix to tubes 1, 3, and 5 and 20 microliters of GMO master mix to tubes 2, 4, and 6. Finally we placed the PCR tubes in the thermal cycler.  On Day 3, we obtained our PCR tube from the thermal cycler and centrifuged it for three seconds. Next we pipetted 10 microliters of Orange G loading dye to test tubes 1-6. Then we loaded 20 microliters of each sample (in the tubes) into rows 1-6 of our gel. In row 7, Mr. Chugh loaded 20 microliters of PCR molecular weight ruler, to act as a control for our lab. Then we ran the gel for 3 minutes at 200 volts. The se stained in Fast Blast DNA stain. The next day, we observed our results.
Results
We concluded that our strawberries were genetically modified.

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Discussion
When observing our gel the next day, we found banding pattern in rows 3, 4, 5 and 6. Because there was a band in lane 4 of the gel, we can conclude that the strawberries we tested were genetically modified. One possible source of error for this lab was not mixing the loading dye well in each sample. Other sources of error could be that PCR or extraction could have been unsuccessful.

GTL: Gene Transformation Lab

Introduction
a) Background- In this lab, we will be using a process known as genetic transformation, where we cause a change in an organism by inserting a gene into an organism. Genetic transformation has been used in many sectors of biotechnology. In farming, it has been used to foster resistance to frost, spoilage, and pests. In bioremediation, bacteria can be transformed to digest oil, which is useful in the cleanup of oil spills. In medicine, diseases caused by malfunctioning genes are now being able to be transformed in gene therapy. This could potentially mean that a person's cells that are making them sick could be transformed with healthy copies.
b) Purpose- The purpose of this lab is to show how by inserting a gene into an organism, we can cause a change in that organism. In this instance, we would be transforming a bacteria with a gene that codes for Green Fluorescent Protein that will cause the bacteria to fluoresce under ultraviolet light.
c) Process- First we are labeling one closed test tube +pGLO and another-pGLO. Then we pipet 250 microliters of transformation solution into the tubs and place them on ice. Using a loop, we pick up the +pGLO immerse the solution at the bottom of the tube and then do the same with -pGLO. Then we add 10 microliters of plasmid DNA directly to the liquid in the +pGLO tube and mix the tube. We incubate the tubes on ice for 10 minutes. While the tubes are on ice, we label four agar plates. Then we heat shock the +pGLO and -pGLO tubes. After we open each tube and add 250 microliters of LB nutrient broth to the tubes and close it. We again incubate the tubes for 10 minutes. After we mix the cubes again and pipet 100 microliters of the transformation and control suspensions onto the appropriate plates. Using sterile loops we spread the suspensions evenly on the surfaces of the plates. Final we stack the plates and tape them together and place the stacks upside down in the 37 degrees Celsius incubator for one day. Then we clean up. 
d) Hypothesis- I hypothesize that the transformed bacteria will glow green under ultraviolet light.
Procedure
Results
Discussion

DNA Chips: Using Microarrays to Discover Diseases

Information
Background: Microarray analysis enables scientists to view and analyze the thousands of genes that have been expresses in cells on one surface. By detecting changes in transcription in cells, one can understand the normal and abnormal functions of a cell. A cancer researcher might look at changes in transcription to learn more about a specific cancer. To do so, a gene chip would be created and imprinted with a pattern of spots which is made up of copies of part of the human genome. The DNA chip would be used to analyze cDNAs that were made from mRNA collected from both cancerous and noncancerous tissue. The tissues would be tagged with dyes and applied to the chip. How much a tissue attaches to its complement on the chip indicates to what extent the transcription occurred. This could help indicate what genes might be important in the development of cancer. The microarray analysis allows suspect genes to be identified sooner due to the technological advances scientists have made. A single microarray chip has more than 30,000 spots of DNA on it. Scientists have learned that some genes are not expressed as much in cancer cells as they are in normal cells. This may indicate how cancer cells spread and grow. Some genes are expressed more in cancer cells than in normal cells, which may play an important role in making cells cancerous. Some genes are also expressed at the same level of both cancer and normal cells. These cells are probably not significant in causing cancer. 
Objective: The objective of this lab is to study the expression of six genes in normal lung cells and lung cancer cells to determine what genes have been transcribed to produce more mRNA in lung cancer cells than in normal lung cells and what genes have been repressed from producing mRNA in the lung cancer cells. Scientists may use this process to discover information about different types of diseases. 
Procedure: We will first prepare the simulated microarray slide. Next, we will obtain DNA tubes from a 70 degrees Celsius water bath. We will set a micropipet to 15 microliters and measure 15 microliter of solution from each of the numbered bottles and place the solution onto the correct spots on the slide using a different tip for each spot. Next, we will drop 15 microliters of hybridization solution to each of the spots. After we will analyze our results and clean up. 
Hypthesis: I think we will use the microarray chip technique to discover which of the six genes causes cancer. 
Procedure
First, we obtained a slide. Next, we set a micropipet to 20 microliters and measured 20 microliters of solution from each numbered bottle and pipetted the solution into the correct spots on the slide, using a different tip for each spot. Nest, we pipetted 20 microliters of "Hybridization Solution" from the dropper bottle onto each spot. After we observed our results. 
Results
Spot 1 turned pink. Spot 2 turned a blue. Spot 3 turned a dark blue-purple color. Spot 4 did not change color. Spot 5 turned light pink. Spot 6 turned a light blue color.
Discussion

Who are you? - Using DNA Profiling in Crime Scene Investigation

Introduction
a. Background- DNA profiling has many applications. Restriction Fragment Length Polymophism (RFLP) is a process of DNA profiling that provides a unique banding pattern based on the restriction sites present in an individuals DNA sequence. They do so by comparing band patterns produced by restriction enzyme cleavage of DNA samples when separated on an agarose gel and is compared to the patterns of potential suspects. Scientists do so by using restriction enzymes, which make cuts at specific sequence of base pairs that is recognizes within the phage DNA, and cut at that site. Bacteria protect their own restriction sites by adding a methyl group. The restriction enzyme sits on a DNA molecule and slides along the helix until it recognizes specific sequences of base pairs that signal the enzyme to stop sliding and cut the DNA molecule at that site. There are more than one restriction site on a DNA molecule and when the restriction enzyme cuts at both of those sites, the result is fragments of different lengths. DNA that has been cut with restriction enzymes can be separated using agarose gel electrophoresis, meaning a current is run through a loaded agarose gel slab. DNA is negatively charged so they are drawn towards the positive pole, so smaller DNA fragments can move through the gel more easily than the larger base pairs. This travel expresses the banding patten in the gel. DNA contains a specific nucleotide sequence, and a radioactive complementary DNA probe can be made that will recognize and bind to that sequence. These probes are used to locate, identify, and compare DNA of different individuals. It is the radioactive tag that allows for the banding pattern to be expressed. Restriction enzymes  are the "chemical scissors" that recognize a particular recognition sequence on DNA, and cut DNA at that point. Two commonly used enzymes are EcoRI and Pstl. Enzymes function best under specific buffer and temperature conditions. In order to make DNA visible, a bluish loading dye is added to the DNA samples. The loading dye does not stain DNA but makes it easier to load the samples and to monitor the progress of the DNA electrophoresis. Bromophenol blue, the "faster" dye, comigrates with DNA fragments 500 bp in a 1% agarose gel, while the xylene cyanol, the "slower" dye, comigrates with DNA fragments 4000 bp in a 1% agarose gel. When the gel is immersed in Fast Blast DNA stain, the stain molecules attract to the DNA trapped in the agarose gel. Two major factors affecting the reliability of DNA fingerprinting are population genetics and genetic statistics. Some segments will show more variation than others.
b. Purpose- DNA typing is used every day to show the relatedness or identity of individual humans, plants, or animals. It is used in industries such as forensics, anthropology, and conservation biology to determine relatedness and to determine teh identity of individuals. It has been used to free innocent suspects in crimes, used in food identification, the reunification of family members and in paternity tests, as well as in identifying human remains. In this particular situation, DNA typing is being used to test and match a suspect to a sample from a crime scene.
c. Summary- In this lab, we will be using restriction enzymes from bacteria to cut DNA at palindromes. It will cut in a specific, staggered way so that sticky ends are created to bind to DNA with an exact shape match. The DNA differences are fragments of different lengths  and then we pipet the DNA into wells in agarose gel. An electric current is run through the gel and the negatively charged DNA is attracted towards the positive end of the gel. It is easier for smaller pieces of DNA to move to the positive end quickly and each DNA sample will provide a specific banding pattern, created by its movement towards the positive end of the gel. We will compare the banding pattern of the crime scene sample to samples of potential suspects in order to narrow the suspects down to one criminal.
d. Hypothesis- I hypothesize that we will be able to match the sample from the crime scene to one of the suspects in order to catch the criminal.
Procedure
On Day One, we first placed the restriction enzyme on ice. We then put 10 micro liters of each DNA sample from the stock tubes and transferred it to the test tubes corresponding with each suspect. We then added 10 micro liters of the enzyme mix into each of the suspect tubes. We then placed the tubes in the centrifuge to mix and collect all the liquid in the bottom of the tube. We then placed the tubes in a water bath of 37 degrees Celsius overnight. The following day, we removed the DNA samples from the refrigerator and centrifuged the tubes again. We then added 5 micro liters of loading dye into each of the tubes and centrifuged it. We then loaded 20 micro liters of the crime scene sample and suspect samples into their respective lanes in the agarose gel. We put the lid on the electrophoresis chamber and turned on the power and ran our samples at 100 V for 3 minutes. When the electrophoresis was complete, we turned off the power and removed the top of the chamber. We then removed the gel and tray and added 120 ml of the 1x Fast Blast DNA stain to the staining tray. we let the gels stain overnight. We then poured out the stain and recorded our results.
Discussion
After examining our results, we determined that the suspect sample in lane 3 turned out to match the sample taken from the crime scene. The banding pattern of sample 3 matched the pattern of the sample from the crime scene. As it turns out, the criminal of the crime was our own lab partner Chloe. There were possible sources of error that could have changed the results of this lab. One possible source of error could have been caused by not using clean pipets when adding the enzyme mix to the samples. It the pipets were not uncontaminated, they could mix the various samples and change the results. 

Fuels of the Future?: Using Enzymes to Create Fuel Alternatives

Introduction
a) Background- Enzymes are proteins that speed up the rate of chemical reactions. Enzyme proteins have an active site where the chemical reaction takes place and the reactant in the reaction is a subtrate, which fits into the active site like a jig-saw puzzle piece as active sites are attracted to the chemicals of the substrate. Increasing the concentration of an enzyme or substrate can change the speed of a reaction. Cellulose is a source of sugar for organisms that is found in the cell walls of plant cells. Cellulose produces a cellulase enzyme family that catalyzes the breaking down of cellulose. Unfortunately, humans and some animals do not contain cellulase while other, plant eating animals do contain the enzyme. In order to make biofuel, fuel created from a biological source that was recently living, creator must covert cellulose into sugar, which is then converted into ethanol by microbial fermentation. Biofuels are a carbon neutral alternative to fossil fuels as it dos not add or take away from the carbon cycle and can be produced sustainably.
b) Purpose- Research companies would apply a lab like this to their own work because it helps identify the most efficient ways to create biofuels. These companies want to create biofuels that do not add to the growing problems our planet faces as we produce greenhouse gas through our exhaustion of the carbon cycle. This lab could find sustainable solutions that would not affect the carbon cycle negatively. The lab is also purposeful to our class as it illustrates that enzymatic reactions are relevant to us.
c) Process- In Day 1, We use enzymes to break down cellulose which leads to the creation of cellobiose. The cellobiose is turned into a usable fuel using cellobiase. Artificial substrate is added to create glucose and P-Nitrophenol. By adding a strong base to kill enzyme, the P-Nitrophenol turns yellow. By adding the strong base, the reaction is slowed down because the enzymes are killed. In Day 2, we grind up mushrooms and add the mushrooms juice to the reactions. The enzymes in the mushroom juice will speed up the reaction because it lowers the activation energy. 
d) Hypothesis- I predict that the reaction will turn more yellow at each time point.
Results
Discussion
When we did this lab, we encountered many problems which involved our results being different than expected. When we were measuring the mushroom extract/substrate mixture to add to each of the cuvettes, we made a mistake, causing us to run out of the mixture sooner than expected. This forced us to only be able to add the mixture to the one minute, two minute, and four minute time points. Despite the mistake on our part, we still were able to see that as the time increased, the solution turned more yellow. These results allowed us to prove our hypothesis correct that the reaction will turn more yellow at each time point.

DNA Precipitation: Capturing Life's Blueprints in a Bottle

Introduction
a) Background- What is DNA? Deoxyribonucleic acid or DNA is a molecule found in almost all cell types and living things. Its purpose is to carry genetic information and is responsible for everything that makes an individual unique. It also has the information for cells to perform all of their functions necessary to survive. Each person has a person combination of DNA, half from your mother and half from your father. DNA at the molecular level is a molecule shaped like a double helix that spirals and has rungs like a ladder, called bases. The bases are adenine, guanine, thymine, and cytosine, and each base functions in a code. Each of the bases are also connected to a sugar and a phosphate, creating nucleotides. When bases are pairing, adenine and thymine always pair and guanine and cytosine also pair. The four bases of DNA make codes that are comprehended by cells called genes, which make proteins that are the basis for almost all the inner workings of the body. The human body has more than 40,000 genes the come together for form the genome. Not all the genes in a cell are used. In the body, all the cells contain the same chromosomes, where the DNA information is located, but different cells read different genes from the chromosomes. It is important to remember that among humans, we share 99.9% identical DNA sequences and it is less that 0.1% sequence variation that distinguishes us from those around us, making humans not so different from one another. DNA does not directly make proteins. The templates for protein synthesis are ribonucleic acid or RNA and messenger RNA or mRNA. It is the job of mRNA to carry the information from the DNA to the ribosomes in a cell the create the protein. The proteins made give cells their traits.
b) Purpose- The purpose of this lab is to extract our own DNA from cheek cells we collect. We extract DNA to get a better look at what DNA looks like and to make it easier to study. This practice can be applied in the real world by scientists who precipitate DNA to study it, map it, compare it, sequence it, clone it, and have other uses for it as well.
c) Process- In order to extract our own DNA, we must first loosen our cheek cells by gently chewing the inside of our cheeks for about 30 seconds. After loosening the cells, we we swished a saline solution (0.9% saltwater) and spit the saline solution containing the skin cells back into the cup which we then transfered into a labeled 15 ml test tube containing 3 ml of water. After we added we added 2 ml of lysis buffer to the tube which breaks open and dissolves the cell membrane which is made of fats and the buffer serves as a detergent to remove the cell membrane. Then we gently invented the tube five times. Next we added 100 ml of protease to the tube to break up protein as well as DNAse which is a protein that kills DNA. The protease contains a salt solution as well, and by adding Na+, we neutralize the DNA and make it both hydrophobic and non polar. After we invert the tube a few times more. Then we place the test tube in a warm water bath at about 50 degrees Celsius to speed up the reaction and break open the cell membrane. Finally we add 5 ml of cold ethanol at a 45 degree angle to ensure that the DNA will not dissolve again. After 5 minutes, we slowly invert the tube five more times to aggregate. Finally, after the DNA is visible, we extracted it and placed it in a necklace. 


Discussion
Although this lab was very informative, we did not have to prove a hypothesis or prove a postulate. However, there were many possible sources of error in the lab. First, we could have not swished the saline solution vigorously enough, therefore not extracting enough cells. When shaking the tubes to aggregate the DNA, we could have inverted the tube too vigorously and could have damaged the DNA precipitation.