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.
Tuesday, October 26, 2010
Tuesday, October 5, 2010
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.
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.
Thursday, September 23, 2010
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.
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.
Wednesday, September 1, 2010
Does bacteria taste good? : Making Yogurt to Prove Koch's Postulates
Introduction
During the 1800's, it was the common belief that diseases could only be transmitted through contact with an infected person. It wasn't until German scientist Robert Koch published his postulate which proved that a certain microbe could cause a certain disease. His first postulate stated that the microorganism would be able to be found in all organisms suffering from the disease but not in healthy organisms. The next postulate stated that the microorganism must be separated and grown in a pure culture. That cultured microorganism should cause the same disease when given to a healthy organism. Finally, the microorganism should be isolated again from to the experimental diseased host and proven to be identical to the original disease-causing microorganism. Because it is considered to be unethical to introduce diseases to humans, we will instead use milk to represent a healthy individual in this lab. We will then prove or disprove the hypothesis that microbes in yogurt are the cause of the "yougurtness disease", or when milk becomes thick and turn into yogurt. We must remember that the majority of the bacteria in yogurt are "probiotic" or bacteria that is beneficial.
To give a little background on bacteria, it is important to note that bacteria has more bacteria on earth than any other life form, making bacteria the most successful species on Earth. Bacteria is classified in the domain of Eukarya and more specifically is a prokaryote as it only consists of one cell, does not have a nucleus, and can not be seen without a microscope. We should also remember that only a small number of bacteria actually cause disease, and the creation of antibiotics has reduced the risk of harmful bacteria. Humans have overused antibiotics though, causing some bacteria to become resistant to antibiotics. Bacteria increase by splitting in half many times logarithmically, which is why it can grow so fast. In order for bacteria to survive, it breaks down sugars using the process of fermentation. Some bacteria also require oxygen in order to survive, and these bacteria are called anaerobes. When yogurt is formed, the yogurt bacteria break down lactose and turn it into pyruvic acid, and then into lactic acid. Lactic acid lowers the pH of milk causing it to curdle and allowing yogurt to stay fresh which still being digestible for people.
In order to make yogurt, you add specific types of bacteria to milk, and allow it ferment in a controlled environment. By doing so, the casein in milk tangles into a solid mass in the denaturation process. This process is due to the increase acidity, which can also allow harmful bacteria to grow. Yogurt in the United States must contain Streptococcis thermophilus and Lactobacillus bulgaricus in order to be sold in stores. In order for a product to be considered yogurt, live bacteria must be present in the product. It is this live yogurt that can be used to inoculate a new batch of yogurt.
Procedure
In order to prove Koch's third postulate, we had to make yogurt. First, we took four test tubes and filled them with a small amount of milk. In test tube 1, our negative control, we added nothing to the tube. In test tube 2, our positive control, we added yogurt to the tube. In test tube 3, we added yogurt and ampicllian (an anti bacteria). In test tube 4, we added E. Coli to the milk. After preparing our test tubes, we placed the tubes in an incubator at 37 degrees Celsius for 24 hours. The following day, we removed the tubes from the incubator and made observation about what happened after the tubes were incubated.
Results/Observations
<object width="480" height="385"><param name="movie" value="http://www.youtube.com/v/Q2bR5DhVrEM?fs=1&hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Q2bR5DhVrEM?fs=1&hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385"></embed></object>
Discussion
After removing the test tubes from the incubator, we noticed many changes in each of the tube. In tube 1, the negative control, the milk curdled overnight and became thick and white. It smelt like fresh milk and had a pH of 6. In tube 2, the milk had also curdled and was a white color, but instead of smelling like fresh milk, it had a tart smell and a pH of 4. The pH was lowered because of lactic acid, a biproduct of the yogurt added to the milk. Lactic acid lowers the pH of milk, making it acidic. In tube 3, the milk remained liquid and was a cloudy white color and had a fresh milk smell with a pH of 7. Although we added yogurt which should have made the milk turn into a thick, white mass after incubation, we also added ampicillin, which ended up killing the yogurt bacteria and leaving the milk. In tube 4, the milk turned into a cloudy liquid and smelt like old milk. It also had a pH of 8. The E. Coli breaks the sugar in the milk down into succinic acid, ethanol, acetic acid, formic acid, and lactic acid, but does not allow the milk to curdle. Although our lab worked, there were many possible sources for error. For example, if we had contaminated any of our tubes, perhaps but using the same loop for getting yogurt and E. Coli, we would have had different results. We also had to make sure to remember that there is bacteria and molds all around us, and if we had touched a contaminant and then touched the tube, there was a chance of contamination.
Procedure
In order to prove Koch's third postulate, we had to make yogurt. First, we took four test tubes and filled them with a small amount of milk. In test tube 1, our negative control, we added nothing to the tube. In test tube 2, our positive control, we added yogurt to the tube. In test tube 3, we added yogurt and ampicllian (an anti bacteria). In test tube 4, we added E. Coli to the milk. After preparing our test tubes, we placed the tubes in an incubator at 37 degrees Celsius for 24 hours. The following day, we removed the tubes from the incubator and made observation about what happened after the tubes were incubated.
Results/Observations
<object width="480" height="385"><param name="movie" value="http://www.youtube.com/v/Q2bR5DhVrEM?fs=1&hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/Q2bR5DhVrEM?fs=1&hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="480" height="385"></embed></object>
Discussion
After removing the test tubes from the incubator, we noticed many changes in each of the tube. In tube 1, the negative control, the milk curdled overnight and became thick and white. It smelt like fresh milk and had a pH of 6. In tube 2, the milk had also curdled and was a white color, but instead of smelling like fresh milk, it had a tart smell and a pH of 4. The pH was lowered because of lactic acid, a biproduct of the yogurt added to the milk. Lactic acid lowers the pH of milk, making it acidic. In tube 3, the milk remained liquid and was a cloudy white color and had a fresh milk smell with a pH of 7. Although we added yogurt which should have made the milk turn into a thick, white mass after incubation, we also added ampicillin, which ended up killing the yogurt bacteria and leaving the milk. In tube 4, the milk turned into a cloudy liquid and smelt like old milk. It also had a pH of 8. The E. Coli breaks the sugar in the milk down into succinic acid, ethanol, acetic acid, formic acid, and lactic acid, but does not allow the milk to curdle. Although our lab worked, there were many possible sources for error. For example, if we had contaminated any of our tubes, perhaps but using the same loop for getting yogurt and E. Coli, we would have had different results. We also had to make sure to remember that there is bacteria and molds all around us, and if we had touched a contaminant and then touched the tube, there was a chance of contamination.
Subscribe to:
Posts (Atom)