At the conclusion of our lab we found that two people in our class were infected with a "disease" that they had unknowingly spread to their classmates after exchanging "bodily fluids". In our lab we began by taking a sample from everyone (these were pre-made by Mr. Chugh) and exchanging them by gently mixing the samples with three people besides ourselves. When we came back the next day after storing the samples at 4 degrees celsius overnight, the found each of our samples.
We loaded each sample into two wells and then added two wells of each positive and negative control to compare our results to. After waitng four minutes we were able to "wash" the samples out with a wash buffer.Then we transferred 50 microliters of antibodies into each well so that the antigens could bind to them. After waiting 4 minutes we washed the samples two more times. After the washing we added 50 microliters of secondary antibody and then waited 4 minutes again, following with three more washes. Lastly we added 50 microliters of enzyme substrate into the twelve wells, this made the contaminated samples turn blue. At our lab table both Elizabeth's and mine were a strong blue, while Ayan and Nate were not contaminated. After surveying the class's results we were able to determine that Taylor Grey and Chloe Krey were the original holders of the "disease.
Sources of error: I shared with four people. My last person being Michael who had just shared with Chloe, leading to my contamination!
Monday, May 23, 2011
Monday, May 16, 2011
Sharing Too Much
In this lab we will be sharing "bodily fluids" with our classmates. We will use a process called ELISA to determine if we have been infected with a contagious disease. ELISA (Enzyme-linked Immunosorbent Assay) is used to detect the presence of a disease agent. When a person is exposed to a "disease agent" their body will react with an immune response. The molecules it uses are called anitgens, within a couple days antibodies ( proteins that recognize the anitgen and bind very tightly) begin circulating in one's body. Anitobodies are made when the body has an immune response.The anitgens are created and then after a period of time the antibodies will begin to specicficallt recognize the anitgen.
The process called ELISA is used for many tests, including pregnancy tests, disease detection, drug testing, testing indoor air quality, and determining if food is labeled correctly. ELISA is used to easily and quickly detect whether patients have been exposed to certain viruses of conditions.
ELISA has four steps:
1. The anitgen is added to the wells of the microplate strip and incubated.
Unbound antigen is washed from the wells with a detergent.
2. Primary anitbody solution is added to the wells and incubated to allow antibody to bind to the antigen.
3. Enzyme- labeled secondary antibody solution is added to the wells.
4. Chromogenic enzyme is added to the wells and incubated to allow color to develope. Then the results are looked at, wells that are colorless are negative and wells that turn blue are positive.
The process called ELISA is used for many tests, including pregnancy tests, disease detection, drug testing, testing indoor air quality, and determining if food is labeled correctly. ELISA is used to easily and quickly detect whether patients have been exposed to certain viruses of conditions.
ELISA has four steps:
1. The anitgen is added to the wells of the microplate strip and incubated.
Unbound antigen is washed from the wells with a detergent.
2. Primary anitbody solution is added to the wells and incubated to allow antibody to bind to the antigen.
3. Enzyme- labeled secondary antibody solution is added to the wells.
4. Chromogenic enzyme is added to the wells and incubated to allow color to develope. Then the results are looked at, wells that are colorless are negative and wells that turn blue are positive.
Wednesday, April 13, 2011
Fish... or not?
In this lab we will be comparing the muscle proteins of many different kinds of fish, determining how closely they are related. We will use protein gel electrophoresis to compare their "fingerprints". By looking at the gel we will be able to determine identify similarities and differences in the fish and their ancestors, or similar species. Each band in the gel will identify a shared characteristic with another type of fish. The muscle proteins of a fish are made up of actin and myosin, along with numerous other muscle tissues.
In this lab we will be grinding up or denaturing the muscle proteins of six different species by putting them in a buffer and then putting them into a 95 degree water bath to denature the proteins, actin and myosin. We will then run the samples on a gel and return the next day to examine the results.
In this lab we will be grinding up or denaturing the muscle proteins of six different species by putting them in a buffer and then putting them into a 95 degree water bath to denature the proteins, actin and myosin. We will then run the samples on a gel and return the next day to examine the results.
Sunday, April 10, 2011
The Passing Along of Mitochondria
In every person there is a set of 46 chromosomes, but in addition to that there is mitochondrial DNA in every human being. Each person's mitochondria has several copies of its own genome. This genome contains 37 genes and is passed to the child through their mother. The mitochondria provides a means to extract energy using oxygen. The mitochondria is in general the same size as bacteria and is circularly shaped.
The mitochondria provides a means to extract energy using oxygen. In this lab, we will extract our DNA the same way we did for the Disease Gene lab, involving saline solution, instagene matrix, and PCR. We will also be using mitochondrial DNA and primers to bracket the mt control region, which will only copy the the specific section of DNA. Next, we will do the same gel electrophoresis that was done in the previous lab to tell us if our mitochondrial DNA is similar.
The mitochondria provides a means to extract energy using oxygen. In this lab, we will extract our DNA the same way we did for the Disease Gene lab, involving saline solution, instagene matrix, and PCR. We will also be using mitochondrial DNA and primers to bracket the mt control region, which will only copy the the specific section of DNA. Next, we will do the same gel electrophoresis that was done in the previous lab to tell us if our mitochondrial DNA is similar.
Thursday, March 24, 2011
Who Is Diseased? No one... That we can tell
Our lab took place over three days. The first day consisted of us making our samples with our own cheek cells, after collecting them in flip-top tubes, we then put them into water baths at 56 degrees Celsius for five minutes, vortexed them, then placed them back in the water bath for a remaining five minutes. After finishing the designated ten minutes, we transported the samples to a 100 degree bath. After five minutes we centrifuged and then put them in the refrigerator until the next day.
The second day of the lab we performed PCR amplification. We retrieved our samples from the refrigerator and then we transferred 20 micro liters of the DNA template from the screw cap tube into the bottom of the PCR tube. We were very careful in not transferring any of the matrix beads because they would have killed the DNA polymerase. We added the yellow master mix to each of our samples and mixed well. Then we added our PCR tubes into the thermal cycler, where it underwent 40 cycles of PCR amplification.
On the third day, we retrieved our tubes and attempted at performing gel electrophoresis. We filled ten lanes of the gel, the first four with "controls" used to compare our results with. The second four lanes had our samples in them. But, unfortunately when we were adding our results one of the samples was being added when the bottom of the gel was punctured and the sample was flooded underneath the gel. Unfortunately, when we got our results back none of our samples showed up except the controls and my own sample (which was the first loaded). So we could not compare our results to our controls to find out who had this diseased gene. However our one sample that did show up, my own, was heterozygous. The way we know this was because after the gel electrophoresis was performed my sample and our heterozygous (+/-) control lined up with my sample.
Although our results did not all show up, we did learn how to test for certain genes and I was able to see how this process could be important to scientists and other people trying to compare information in different genes.
The second day of the lab we performed PCR amplification. We retrieved our samples from the refrigerator and then we transferred 20 micro liters of the DNA template from the screw cap tube into the bottom of the PCR tube. We were very careful in not transferring any of the matrix beads because they would have killed the DNA polymerase. We added the yellow master mix to each of our samples and mixed well. Then we added our PCR tubes into the thermal cycler, where it underwent 40 cycles of PCR amplification.
On the third day, we retrieved our tubes and attempted at performing gel electrophoresis. We filled ten lanes of the gel, the first four with "controls" used to compare our results with. The second four lanes had our samples in them. But, unfortunately when we were adding our results one of the samples was being added when the bottom of the gel was punctured and the sample was flooded underneath the gel. Unfortunately, when we got our results back none of our samples showed up except the controls and my own sample (which was the first loaded). So we could not compare our results to our controls to find out who had this diseased gene. However our one sample that did show up, my own, was heterozygous. The way we know this was because after the gel electrophoresis was performed my sample and our heterozygous (+/-) control lined up with my sample.
Although our results did not all show up, we did learn how to test for certain genes and I was able to see how this process could be important to scientists and other people trying to compare information in different genes.
Wednesday, March 16, 2011
Who Is Diseased?
In this lab, we will be using PCR to examine our own DNA sequence in a test tube. We will be looking for a particular piece of DNA that is present in most people, but not all people. We will be determining this through PCR, which has begun transforming molecular biology into a multidisciplinary research field. On the first day we will be extracting DNA from our cheek cells. We will break open the cell membranes by putting the samples into a water bath, then we will use instagene matrix beads to kill DNase. On day two we will perform PCR in three steps, as shown below....
In this lab PCR has three main steps. The first is denaturation, the second is an annealing step, and the third, an extension step. In the first step, the DNA seperates into two single stranded molecules. The templates must be sperated before the polymerase can generate a copy.
During the annealing step the oligonucleotide primers find their complementary sequenceds on two single stranded template strands of DNA. After they are annealed they can be used as primers for the Taq DNA polymerase. In extension Taq DNA polymerase's job is too add nucleotides (A,T, G and C) to the primer to create a complementary copy of the DNA template.
After running through this, on day three we will use gel eletrophoresis to diagram our results.
In this lab PCR has three main steps. The first is denaturation, the second is an annealing step, and the third, an extension step. In the first step, the DNA seperates into two single stranded molecules. The templates must be sperated before the polymerase can generate a copy.
During the annealing step the oligonucleotide primers find their complementary sequenceds on two single stranded template strands of DNA. After they are annealed they can be used as primers for the Taq DNA polymerase. In extension Taq DNA polymerase's job is too add nucleotides (A,T, G and C) to the primer to create a complementary copy of the DNA template.
After running through this, on day three we will use gel eletrophoresis to diagram our results.
Thursday, February 10, 2011
GMO's results
After learning all about genetically modified organisms over the past week from Mr.Chugh, we have finally finished our three day lab and have found results. Each lab table was able to bring in a fruit or vegetable to test to see if it was genetically modified. On the first day we extracted the DNA from our food samples (our control and test foods). We measured out about one gram of each substance and then added water into a mortar where we used a pestle to form a "slurry". Once we had ground the substance smooth enough we pipetted it into a test tube. We then added our test tubes to a water bath at 99 degrees Celsius for five minutes, followed by centrifuging for five minutes. That completed our first day of extracting genetic material from our test food.
On the second day, we returned and added "master mix" to each sample. After adding it to each PCR tube we put it in the thermal cycler. The cycler goes through three cycles. The first is at about 95 degrees Celsius, this is where denaturization occurs (DNA is unravelled), the second temperature change is to about 60 degrees Celsius where the primer binds, and the last stage is about 72 degrees where DNA polymerase appears.
On the third day of this lab, where we did gel electrophoresis. After retrieving our PCR tubes, we added loading dye to each sample and mixed them. Next, we added 20 micro liters of each sample into the gel. After loading them in we ran an agarose gel for 3 minutes on 200 V.
When we returned on the fourth day, we found our results! Our food (broccoli) was organic, meaning it was not genetically modified! After evaluating our electrophoresis we found bands in the second, fourth and sixth lanes. These were the lanes that had GMO primers added to them, meaning they would show up in our testing, unlike the plant primers because the plant primers were looking for non-modified genes to code for. The GMO primers were looking for modified genes because it can only copy them if it recognizes the genes.
Possible causes for error: Our results could have been more accurate had the experiment been done in a sterilized lab with better pipetting and more precise timing with water baths.
http://www.youtube.com/watch?v=dvlpCxjxC1g (picture is a little blurry)
On the second day, we returned and added "master mix" to each sample. After adding it to each PCR tube we put it in the thermal cycler. The cycler goes through three cycles. The first is at about 95 degrees Celsius, this is where denaturization occurs (DNA is unravelled), the second temperature change is to about 60 degrees Celsius where the primer binds, and the last stage is about 72 degrees where DNA polymerase appears.
On the third day of this lab, where we did gel electrophoresis. After retrieving our PCR tubes, we added loading dye to each sample and mixed them. Next, we added 20 micro liters of each sample into the gel. After loading them in we ran an agarose gel for 3 minutes on 200 V.
When we returned on the fourth day, we found our results! Our food (broccoli) was organic, meaning it was not genetically modified! After evaluating our electrophoresis we found bands in the second, fourth and sixth lanes. These were the lanes that had GMO primers added to them, meaning they would show up in our testing, unlike the plant primers because the plant primers were looking for non-modified genes to code for. The GMO primers were looking for modified genes because it can only copy them if it recognizes the genes.
Possible causes for error: Our results could have been more accurate had the experiment been done in a sterilized lab with better pipetting and more precise timing with water baths.
http://www.youtube.com/watch?v=dvlpCxjxC1g (picture is a little blurry)
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