Part 1: Introduction
This lab will let us test if we have a contagious "disease." The ELISA method tests for the presence of antibodies specific to a disease. So if you have the disease, the antibodies will be present, and if you don't, then the antibodies will be absent. This method has had a huge impact on the medical world. It is currently commonly used for disease detection, pregnancy tests, detecting drug use, testing air quality, and determining if food is actually what it's labeled.
Part 2: Experiment
First, we will label a yellow tube and pipet with our initials. Then we'll transfer our bodily fluid sample into another student's tube, then mix the samples, and take back half (750 microliters) and put it in our own tube, and write down their name and the time it was collected (including seconds.) Then we'll repeat the sharing protocol two more times when instructed, and discard the pipet. Then we'll store the tubes overnight.
On the next day, we'll label our 12-well strip as indicated in the manual. Then we'll use a fresh pipet tip to transfer 50 microliters of the positive control into the 3 positive wells, and use another fresh pipet tip to put 50 microliters of the negative control into the 3 negative wells. Then we'll wait 5 minutes, then wash the microplate strip on the paper towels. Then we'll use a new transfer pipet to fill each well with wash buffer, then drain it again on the paper towels. Then we'll do this washing step again. After that we'll transfer 50 microliters of primary antibody into all 12 wells, then wait 5 minutes, and wash twice again. Next we'll use a new pipet tip to transfer 50 microliters of secondary antibody into all 12 wells, awit 5 minutes, and wash 3 times. Then using a new pipet tip, we'll put 50 microliters of enzyme substrate into all 12 wells. Then we'll wait 5 minutes, and observe and record the results. Then we clean up, and we're done!
Monday, May 16, 2011
Monday, April 11, 2011
Hands-On Proteomics
Part 1: Introduction
Proteonomics, the study of the structures and functions of proteins, is perceived as the "next step" in research, the last step being genomics. The proteome, unlike the genome, differs from cell to cell and constantly changes, so it is fairly difficult to study. Some research suggests that a lot of noncoding DNA in the genome functions to regulate protein production, expression levels, and modifications to proteins after translation, and that these variations are what differentiate humans from other species with similar genomes. We have also learned that one gene can code for many proteins and mechanisms that finely regulate things like cellular locations and expression levels of different proteins. In this lab, we will run several proteins on a gel and determine if they are of similar species.
Part 2: Experiment
First, we willl label one fliptop micro tube and also one screwcap micro tube for each of five fish samples. Then we'll add 250 microliters of Laemmli sample buffer to each fliptop tube. Then we'll cut a small piece of fish muscle from each sample and put it in the appropriate fliptop tube, then flick the tubes to mix it up. Then we'll incubate the tubes for 5 minutes at room temperature. Next we'll pour only the buffer from each fliptop tube into the corresponding screwcap tube. We will heat the screwcap tube samples for 5 minutes at 95 C.
On the next day, we'll set up a Mini-Protean 3 gel box and add electrophoresis buffer to the chamber. We'll prepare the gel as indicated in the lab guide. Then we'll put it in the mini tank, and fill it with electrophoresis buffer. Meanwhile we will have heated the fish samples and actin and myosin standards to 95 C for 2-5 minutes. Then we'll load the gels as indicated in the lab guide, and electrophorese for 30 minutes at 200 volts. Then we'll remove the gels, rinse them off, and stain the gels for at least 1 hour. Then we'll destain the gels in water overnight.
On the third day, we'll dry the gels using GelAir cellophane.
Proteonomics, the study of the structures and functions of proteins, is perceived as the "next step" in research, the last step being genomics. The proteome, unlike the genome, differs from cell to cell and constantly changes, so it is fairly difficult to study. Some research suggests that a lot of noncoding DNA in the genome functions to regulate protein production, expression levels, and modifications to proteins after translation, and that these variations are what differentiate humans from other species with similar genomes. We have also learned that one gene can code for many proteins and mechanisms that finely regulate things like cellular locations and expression levels of different proteins. In this lab, we will run several proteins on a gel and determine if they are of similar species.
Part 2: Experiment
First, we willl label one fliptop micro tube and also one screwcap micro tube for each of five fish samples. Then we'll add 250 microliters of Laemmli sample buffer to each fliptop tube. Then we'll cut a small piece of fish muscle from each sample and put it in the appropriate fliptop tube, then flick the tubes to mix it up. Then we'll incubate the tubes for 5 minutes at room temperature. Next we'll pour only the buffer from each fliptop tube into the corresponding screwcap tube. We will heat the screwcap tube samples for 5 minutes at 95 C.
On the next day, we'll set up a Mini-Protean 3 gel box and add electrophoresis buffer to the chamber. We'll prepare the gel as indicated in the lab guide. Then we'll put it in the mini tank, and fill it with electrophoresis buffer. Meanwhile we will have heated the fish samples and actin and myosin standards to 95 C for 2-5 minutes. Then we'll load the gels as indicated in the lab guide, and electrophorese for 30 minutes at 200 volts. Then we'll remove the gels, rinse them off, and stain the gels for at least 1 hour. Then we'll destain the gels in water overnight.
On the third day, we'll dry the gels using GelAir cellophane.
Monday, March 28, 2011
Mitochondrial DNA Amplification
Part 1: Introduction
Aside from normal cellular DNA, there is a type called "mitochondrial DNA," or mtDNA. It contains only 37 genes, instead of 46. The purpose of this DNA is to code for mitochondria to produce energy and store it in the form of ATP.
This is very significant, because using this mtDNA, we can trace back lineage -- as it is only inherited by the mother -- to many generations back. Scientists are believed to have used this technology to trace the "mitochondrial Eve," the common ancestor of the modern human race, in Africa about 200,000 years ago. This technology is also useful for creating family trees, identifying family ties, and identifying remains.Mitochondrial DNA is the easiest to extract, as it is very, very amplified already in each cell, so it can even be found in dead cells; a lot more easily than non-mitochondrial cellular DNA.
Part 2: Experiment
In this lab, we will be finding our own mitochondrial DNA. The process is almost exactly the same as the last one, so for detailed instructions, please refer to the last lab intro. The difference between the last one and this one is that we will be using different primers to find the mtDNA.
Aside from normal cellular DNA, there is a type called "mitochondrial DNA," or mtDNA. It contains only 37 genes, instead of 46. The purpose of this DNA is to code for mitochondria to produce energy and store it in the form of ATP.
This is very significant, because using this mtDNA, we can trace back lineage -- as it is only inherited by the mother -- to many generations back. Scientists are believed to have used this technology to trace the "mitochondrial Eve," the common ancestor of the modern human race, in Africa about 200,000 years ago. This technology is also useful for creating family trees, identifying family ties, and identifying remains.Mitochondrial DNA is the easiest to extract, as it is very, very amplified already in each cell, so it can even be found in dead cells; a lot more easily than non-mitochondrial cellular DNA.
Part 2: Experiment
In this lab, we will be finding our own mitochondrial DNA. The process is almost exactly the same as the last one, so for detailed instructions, please refer to the last lab intro. The difference between the last one and this one is that we will be using different primers to find the mtDNA.
Tuesday, March 15, 2011
Genetic Defect Lab
Part 1: Introduction
In this lab, we will be using a Polymerase Chain Reaction to see if a certain pattern of DNA is present in our cells. PCR has a wide range of uses, such as gene mapping, cloning, DNA sequencing, and the one we will be using, gene detection. The way this is done is by taking a DNA sample, and amplifying the gene of interest through PCR.
Part 2: Experiment
On the first day, we will chew our cheeks and rinse our mouths with a saline solution and put it in a cup. Then we'll transfer 1ml of it into a micro test tube with 200 microliters of InstaGene matrix. Then we'll spin it in a centrifuge for 2 minutes. Then we'll pour out the saline solution while keeping the DNA pellet at the bottom of the tube, and flick the tube to resuspend the pellet. Then we'll put all of the resuspended cells into the screwcap tube with InstaGene using a 20 microliter micropipet, and throw out the flip-top tube that formerly contained the pellet of DNA. Then we'll shake or vortex the screwcap tube. When we're done, we'll put our tubes in a waterbath at 56 C for 10 minutes, shaking them after 5 minutes, then putting them back. Then we'll put the tubes in a 100 C waterbath for 5 minutes. Then we'll vortex the tube for a couple minutes and put it in the fridge until day 2.
On the second day, we'll get our screwcap tube and spin it in a centrifuge. Then with a 20 microliter pipette, we'll extract part of the "supernatant," which is the liquid above the DNA pellet.Then we'll put 20 microliters of the yellow master mix into the PCR tube and mix it. Then we'll put the PCR tube in the thermal cycler for 40 cycles. We'll make sure to label our tubes and record their location in the cycler.
On the third day, we will get our PCR tube and centrifuge it for 3 seconds. Then we'll add 10 microliters of PV92 XC loading dye and mix it gently. Then we'll cover the gel on the electrophoresis apparatus with 1x TAE buffer; about 275 mL of it. Then we'll load the lanes with the samples indicated in the packet. Then we will run the electrophoresis process at 200v for 3 minutes or 12 minutes, depending on period. Then we'll put the gel into the plastic staining tray and await further instructions.
Part 3: Discussion
In this lab, we will be using a Polymerase Chain Reaction to see if a certain pattern of DNA is present in our cells. PCR has a wide range of uses, such as gene mapping, cloning, DNA sequencing, and the one we will be using, gene detection. The way this is done is by taking a DNA sample, and amplifying the gene of interest through PCR.
Part 2: Experiment
On the first day, we will chew our cheeks and rinse our mouths with a saline solution and put it in a cup. Then we'll transfer 1ml of it into a micro test tube with 200 microliters of InstaGene matrix. Then we'll spin it in a centrifuge for 2 minutes. Then we'll pour out the saline solution while keeping the DNA pellet at the bottom of the tube, and flick the tube to resuspend the pellet. Then we'll put all of the resuspended cells into the screwcap tube with InstaGene using a 20 microliter micropipet, and throw out the flip-top tube that formerly contained the pellet of DNA. Then we'll shake or vortex the screwcap tube. When we're done, we'll put our tubes in a waterbath at 56 C for 10 minutes, shaking them after 5 minutes, then putting them back. Then we'll put the tubes in a 100 C waterbath for 5 minutes. Then we'll vortex the tube for a couple minutes and put it in the fridge until day 2.
On the second day, we'll get our screwcap tube and spin it in a centrifuge. Then with a 20 microliter pipette, we'll extract part of the "supernatant," which is the liquid above the DNA pellet.Then we'll put 20 microliters of the yellow master mix into the PCR tube and mix it. Then we'll put the PCR tube in the thermal cycler for 40 cycles. We'll make sure to label our tubes and record their location in the cycler.
On the third day, we will get our PCR tube and centrifuge it for 3 seconds. Then we'll add 10 microliters of PV92 XC loading dye and mix it gently. Then we'll cover the gel on the electrophoresis apparatus with 1x TAE buffer; about 275 mL of it. Then we'll load the lanes with the samples indicated in the packet. Then we will run the electrophoresis process at 200v for 3 minutes or 12 minutes, depending on period. Then we'll put the gel into the plastic staining tray and await further instructions.
Part 3: Discussion
Thursday, February 3, 2011
GM Food Testing Lab
Part 1: Introduction
About 70% of food sold in supermarkets are now genetically modified. This is a touchy subject for many. A lot of people don't like the fact that their food isn't all-natural, but does that really make sense? Genetic modification allows produce to stay fresh longer, makes them invulnerable to certain pests and pesticides, and makes them grow larger and faster. But some people not sold on the idea of eating genetically modified foods. The debate remains heated, but in this lab, we will take a sample of fruit from a supermarket and test to see if it is indeed genetically modified or not.
Part 2: Experiment
(Note that this procedure summary will be slightly abridged, as it is a three-day lab with many steps.)
On the first day, we'll extract the DNA from our apple and corn flour. This will be done with a mortar and pestle; one for each food sample. We will crush the matter as much as we can to break the cell walls, adding 5 mL of water for each gram of food. We have 1.86g of apple and 0.86g of corn flour. Then we'll take 50 microliters of each solution and put them into their respective tubes of InstaGene solution to solidify the DNA. We'll then put the tubes in a waterbath for 5 minutes, then centrifuge them for 5 minutes, then put them in the fridge overnight.
On the second day, we'll get 6 new tubes and label them 1-6, with contents according to the table in the packet. We'll put them all in bigger capless tubes and put them in a foam block and float them in ice water. We'll add 20 microliters of each indicated master mix to each tube, using a new tip for each one, and cap the tubes. Then we'll add 20 microliters of each indicated DNA type to each tube, while using a new tip again. We won't want to mix up the InstaGene pellet at the bottom. Then we'll gently mix the tubes, again without mixing up the InstaGene pellet, and then recap the tubes, and put them in the thermal cycler.
On the third day, we'll do the electrophoresis of the tubes. We'll get our tubes from the cycler and put the PCR tube in the capless tube, and pulse-spin it for 3 seconds. With a fresh tip, we'll add 10 microliters of orange G loading dye to each sample and mix them well. Then we'll add 20 microliters of the molecular weight ruler and 20 microliters of each sample into our gel in the order indicated in the packet. Then we'll run the gel for the time and voltage depending on what gel we're using. Then we'll stain the gels with Fast Blast DNA stain. After that, we'll analyze our results and see what happened.
About 70% of food sold in supermarkets are now genetically modified. This is a touchy subject for many. A lot of people don't like the fact that their food isn't all-natural, but does that really make sense? Genetic modification allows produce to stay fresh longer, makes them invulnerable to certain pests and pesticides, and makes them grow larger and faster. But some people not sold on the idea of eating genetically modified foods. The debate remains heated, but in this lab, we will take a sample of fruit from a supermarket and test to see if it is indeed genetically modified or not.
Part 2: Experiment
(Note that this procedure summary will be slightly abridged, as it is a three-day lab with many steps.)
On the first day, we'll extract the DNA from our apple and corn flour. This will be done with a mortar and pestle; one for each food sample. We will crush the matter as much as we can to break the cell walls, adding 5 mL of water for each gram of food. We have 1.86g of apple and 0.86g of corn flour. Then we'll take 50 microliters of each solution and put them into their respective tubes of InstaGene solution to solidify the DNA. We'll then put the tubes in a waterbath for 5 minutes, then centrifuge them for 5 minutes, then put them in the fridge overnight.
On the second day, we'll get 6 new tubes and label them 1-6, with contents according to the table in the packet. We'll put them all in bigger capless tubes and put them in a foam block and float them in ice water. We'll add 20 microliters of each indicated master mix to each tube, using a new tip for each one, and cap the tubes. Then we'll add 20 microliters of each indicated DNA type to each tube, while using a new tip again. We won't want to mix up the InstaGene pellet at the bottom. Then we'll gently mix the tubes, again without mixing up the InstaGene pellet, and then recap the tubes, and put them in the thermal cycler.
On the third day, we'll do the electrophoresis of the tubes. We'll get our tubes from the cycler and put the PCR tube in the capless tube, and pulse-spin it for 3 seconds. With a fresh tip, we'll add 10 microliters of orange G loading dye to each sample and mix them well. Then we'll add 20 microliters of the molecular weight ruler and 20 microliters of each sample into our gel in the order indicated in the packet. Then we'll run the gel for the time and voltage depending on what gel we're using. Then we'll stain the gels with Fast Blast DNA stain. After that, we'll analyze our results and see what happened.
Thursday, January 27, 2011
Glowing Bacteria Lab
Part 1: Introduction
In this lab, we'll be performing what is known as "genetic transformation," which is the modification of genes in an organism. In the biotechnology industry, this is used in many applications, like genetically modified foods, bioremediation (using bacteria to digest oil spills,) and gene therapy in people with defective genes. We will be taking a Green Fluorescent Protein (GFP)-producing gene from glowing jellyfish, and inserting it into bacteria, which will incorporate the gene into its DNA, and produce the same glowing effect, and pass the trait on to its offspring.
Part 2: Experiment
First we'll label both of our tubes: one as +pGLO and the other -pGLO, and our table number on both, then put them in the foam block. Then we'll put 250 microliters of transformation solution (calcium chloride) into each tube with a sterile pipet, then ice the tubes. Next we'll get a colony of bacteria from our starter plate with a sterile loop, and it put it in the +pGLO tube, then put the tube back on the ice. Then we'll repeat the process with the -pGLO tube, with a new sterile loop. Next we'll add 10 microliters of plasmid DNA to the +pGLO tube only, and flick the tube to mix it. Then we'll put the tubes on ice and let them sit for 10 minutes. While that's going on, we'll label our 4 agar plates. Next we'll do a heat shock treatment by taking the tubes from the ice to a 42 C waterbath for 50 seconds, then back onto the ice for 2 minutes. Then we'll add 250 microliters of LB broth to each tube, one at a time, with a sterile pipet. We'll then let the tubes sit at room temperature for 10 minutes. Next we'll mix the tubes by tapping them with a finger, and with a new sterile pipet for each tube, we'll pipet 100 microliters of the liquid onto the corresponding plates. Next, using a new sterile loop for each plate, we'll spread the liquid around the surface of the plate. Finally, we'll stack our plates, tape them together, put our table number on the bottom of the stack, and put the stack upside down in the 37 C incubator until Monday.
Part 3: Results
Sure enough, the expected results of the lab were successful, and the bacteria glowed as expected. Since htis operation worked, I can't think of any possible margin of error, as there aren't any other stray glowing genes around that could have contaminated the sample. Our only slip-up was that the trays were left in the cold room all weekend, so the bacteria couldn't grow very quickly, and some probably died, but we did have some surviving glowing bacteria. This is probably my favorite lab we have done all year because of the level of technological advancement it uses-- it is the most current and relevant one that we have done in my opinion.
In this lab, we'll be performing what is known as "genetic transformation," which is the modification of genes in an organism. In the biotechnology industry, this is used in many applications, like genetically modified foods, bioremediation (using bacteria to digest oil spills,) and gene therapy in people with defective genes. We will be taking a Green Fluorescent Protein (GFP)-producing gene from glowing jellyfish, and inserting it into bacteria, which will incorporate the gene into its DNA, and produce the same glowing effect, and pass the trait on to its offspring.
Part 2: Experiment
First we'll label both of our tubes: one as +pGLO and the other -pGLO, and our table number on both, then put them in the foam block. Then we'll put 250 microliters of transformation solution (calcium chloride) into each tube with a sterile pipet, then ice the tubes. Next we'll get a colony of bacteria from our starter plate with a sterile loop, and it put it in the +pGLO tube, then put the tube back on the ice. Then we'll repeat the process with the -pGLO tube, with a new sterile loop. Next we'll add 10 microliters of plasmid DNA to the +pGLO tube only, and flick the tube to mix it. Then we'll put the tubes on ice and let them sit for 10 minutes. While that's going on, we'll label our 4 agar plates. Next we'll do a heat shock treatment by taking the tubes from the ice to a 42 C waterbath for 50 seconds, then back onto the ice for 2 minutes. Then we'll add 250 microliters of LB broth to each tube, one at a time, with a sterile pipet. We'll then let the tubes sit at room temperature for 10 minutes. Next we'll mix the tubes by tapping them with a finger, and with a new sterile pipet for each tube, we'll pipet 100 microliters of the liquid onto the corresponding plates. Next, using a new sterile loop for each plate, we'll spread the liquid around the surface of the plate. Finally, we'll stack our plates, tape them together, put our table number on the bottom of the stack, and put the stack upside down in the 37 C incubator until Monday.
Part 3: Results
Sure enough, the expected results of the lab were successful, and the bacteria glowed as expected. Since htis operation worked, I can't think of any possible margin of error, as there aren't any other stray glowing genes around that could have contaminated the sample. Our only slip-up was that the trays were left in the cold room all weekend, so the bacteria couldn't grow very quickly, and some probably died, but we did have some surviving glowing bacteria. This is probably my favorite lab we have done all year because of the level of technological advancement it uses-- it is the most current and relevant one that we have done in my opinion.
Wednesday, November 17, 2010
Identifying Essential Genes
Part 1: Introduction
The analysis of the human genome was first called pointless and a "waste of time" by critics, but as research has been performed and the genome has been collected, we have found many ways of applying our genetic sequences to very significant life scenarios. The human genome has been used to identify disease-causing genes, develop pharmaceuticals for diseases, study evolutionary patterns across species, track generational genetic lineage among humans, identify gene functions, and figure out how the genome works in general. In this lab, we'll use a small-scale simulation of a microarray to test 6 genes that are present in lung cells, and test for differences between these genes in healthy lung cells and cancerous lung cells.
Part 2: Experiment
First we'll get our 2 slides. Each have 8 spots for aqueous solutions, though we'll only use 6, since we're only testing for 6 genes. We'll put 20 microliters of each gene sequence on a corresponding spot-- gene 1 on spot 1, gene 2 on spot 2, etc. Then we'll put 20 microliters of a cDNA solution, made with cDNA from each lung tissue sample, on each spot. After about a minute, we'll observe the spots for our results. Blue dots will represent a gene expressed in only the healthy lung sample, pink dots will represent a gene expressed only in the cancerous lung sample, clear dots will represent that the gene isn't in either, and purple will represent that the gene is represented in both.
Part 3: Discussion
We found that the genes expressed in the lung cancer cells (pink) were C4BPA, SIAT9, and partially ODC1. Genes expressed in the healthy lung cells (blue) were partially ODC1, FGG, and CYP24. ODC1 was expressed in both, so it was normal in both samples; it's possible that it's a gene that synthesizes proteins for the lungs. HBG1 was expressed in neither sample. It codes for hemoglobin, which is in blood, which is not specifically directed or produced in the lungs. C4BPA or SIAT9 may be responsible for the cancer in the lung cells, as they are not present in healthy ones, but are present in the cancerous cells.
Some possible sources of error could have been contamination between specimens, dirty slides, incorrect measurements, or user error in placing genes in correct spots.
The analysis of the human genome was first called pointless and a "waste of time" by critics, but as research has been performed and the genome has been collected, we have found many ways of applying our genetic sequences to very significant life scenarios. The human genome has been used to identify disease-causing genes, develop pharmaceuticals for diseases, study evolutionary patterns across species, track generational genetic lineage among humans, identify gene functions, and figure out how the genome works in general. In this lab, we'll use a small-scale simulation of a microarray to test 6 genes that are present in lung cells, and test for differences between these genes in healthy lung cells and cancerous lung cells.
Part 2: Experiment
First we'll get our 2 slides. Each have 8 spots for aqueous solutions, though we'll only use 6, since we're only testing for 6 genes. We'll put 20 microliters of each gene sequence on a corresponding spot-- gene 1 on spot 1, gene 2 on spot 2, etc. Then we'll put 20 microliters of a cDNA solution, made with cDNA from each lung tissue sample, on each spot. After about a minute, we'll observe the spots for our results. Blue dots will represent a gene expressed in only the healthy lung sample, pink dots will represent a gene expressed only in the cancerous lung sample, clear dots will represent that the gene isn't in either, and purple will represent that the gene is represented in both.
Part 3: Discussion
We found that the genes expressed in the lung cancer cells (pink) were C4BPA, SIAT9, and partially ODC1. Genes expressed in the healthy lung cells (blue) were partially ODC1, FGG, and CYP24. ODC1 was expressed in both, so it was normal in both samples; it's possible that it's a gene that synthesizes proteins for the lungs. HBG1 was expressed in neither sample. It codes for hemoglobin, which is in blood, which is not specifically directed or produced in the lungs. C4BPA or SIAT9 may be responsible for the cancer in the lung cells, as they are not present in healthy ones, but are present in the cancerous cells.
Some possible sources of error could have been contamination between specimens, dirty slides, incorrect measurements, or user error in placing genes in correct spots.
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