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

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.

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.

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.

DNA Fingerprinting - Identification of the Future

 Part 1: Introduction 
DNA, the code of our lives, can now be extracted from animal cells and read like a personal ID card. This is a recent breakthrough that has been revolutionizing the scientific community. It's most commonly known as the main identification method in forensics, to find the truth of the locations of certain people at certain times. It has also been used in food identification, to find out if certain meats are actually pure meat of that type, or if something different is mixed in, and if the food is an endangered species. Also with animals, seeing the genetic code of certain species can determine whether the species are related or not. DNA testing has also cleared falsely convicted criminals of charges, and even saved falsely accused people from death row. DNA is also used to identify corpses if their teeth or fingerprints are compromised, or if only parts of the body remain. DNA testing is the light of truth where before there was none.
In this lab, we'll be comparing DNA samples to simulated criminals' DNA to find the perpetrator. This specific method is used in forensics, but slight variations of it apply to all the uses of DNA above.

  Part 2: Experiment
For the procedure, we'll have 6 tubes-- 1 of the "crime scene" DNA, and 1 for each of the 5 suspects. We'll add a restriction enzyme to each one to break apart the DNA at very specific places. We'll then put the tubes in a microcentrifuge to make sure the solution is mixed well. Then we'll put the tubes in a waterbath at 37C overnight. The next day, we'll place them in an ice block. Then we'll put the tubes in the microcentrifuge again to collect all the liquid at the bottom of the tube. Then we'll add loading dye to each tube so we can see approximately where the DNA is, and the dye also keeps the DNA sample in the wells of the gel. After putting in the dye we'll mix it in the microcentrifuge again. Then we put one sample in each lane of the agarose gel, which is inside the electrophoresis apparatus, and use a DNA size marker control for the first lane. Then we'll do the electrophoresis process. After that we'll take out the gel and put it in a staining tray, then put DNA stain in the tray (enough to cover the gel.) The next day, we'll look at the gel and record our results.

 Part 3: Discussion
 We found the perpetrator to be Katie, because the pattern made by her DNA matched the pattern of the crime scene DNA. One possible source of error could have been contamination, crossover of DNA solution in the gel, mislabeling a tube, or hearing another group's results.

Biofuels - Making Something Useful Out of Green Waste

Part 1: Introduction  

Biofuels have long been a topic of interest, though much more lately as we entered the "green" trend. As global warming becomes apparent, and as the price of gas rises, more and more people are looking to buy alternative cars, and alternative sources of energy to power their appliances. This demand for green technology has scientists scrambling to find an efficient way to recycle energy that won't harm the environment. Biofuels are usually combustible fuels that are made from biomass. They can be made by adding enzymes to plant material to break them down and release the energy stored inside them.
In this lab, we'll be making a biofuel out of cellulose by adding enzymes, and we'll measure how much fuel is made at several time intervals. My hypothesis is that there will be more indication of fuel as time progresses. We'll have tubes of non-enzyme solutions as controls.

Part 2: Experiment

 First, we'll get 7 vials, and label each one: Start, End, E1, E2, E3, E4, and E5. We'll put 500 microliters of stop solution into each one. Then we'll label one 15 ml conical tube "Enzyme Reaction" and the other "Control." Next we'll put 2 ml of 1.5 mM substrate into the "Enzyme Reaction" tube, then 1 ml of the same solution into the "Control" tube. Then we'll label two DPTP's, one "E" for Enzyme, and one "C" for Control.

After preparation, the reaction starts. We use the "C" pipet and put 500 microliters of buffer into the Control tube and mix gently, then we'll remove 500 microliters of the solution and put it into the "Start" vial. Then we'll take the "E" pipet and put 1 ml of enzyme into the Enzyme Reaction tube, and gently mix, then start our timer. At several time stops-- 1 min, 2 min, 4 min, 6 min, and 8 min-- we'll remove 500 microliters of the Enzyme Reaction tube and add it to each vial (e.g. at 1 min, 500 microliters into E1. at 2 min, 500 microliters into E2. etc). After these are all done, we'll use the "C" pipet and take out 500 microliters from the Control tube and put it in the "End" vial. Then we'll wash everything out.

The next day, we'll get a mushroom, measure out 1 g of it, put it into a mortar, add 2 ml of extraction buffer, and make mush with the pestle. Then we'll strain out the solid particles and put the juice in a 1.5 ml microcentrifuge tube. We'll put 500 microliters of stop solution into each of 6 vials (now clean). Then we'll label a 15 ml tube as the type of mushroom, and then put 3 ml of substrate into the tube. Next, we'll put 250 microliters of the mushroom juice into the tube of substrate, and start the timer. At the same time intervals as last time, we'll take out 500 microliters of mushroom extract/substrate mixture from the reaction tube and add it to each of only 5 vials. Then we'll add 500 microliters of extraction buffer to vial 6, and then add one drop of mushroom juice to it. Then we'll analyze the results.

Part 3: Discussion
 
In each tube through the experiment, each one got progressively more yellow in the slightest bit. It was extremely hard to notice. I had thought it would be a more apparent change.
I can't think of any possible sources of error, except for the tubes being possibly not all the way washed out, and the samples not being pure, as we didn't measure out the supply tubes; only the test ones.

Extracting Our Own DNA - And Wearing It

Part 1: Introduction  
 
DNA is a long, double-stranded helix nucleic acid contained in the nucleus of all animal cells. Its purpose is to store all the genetic information of the organism. DNA is comprised of nucleotides, which are simple molecules formed by 3 substances: a sugar (ribose or deoxyribose), a phosphate, and 1 of 4 nitrogenous bases (adenine, thymene, cytosene, guanine.) These bases are what code the genetic information used to determine almost all of the organism's characteristics. The correct formation and function of DNA is incredibly important for any organism, as a flaw can prove deadly. Genetic disfunctions can kill an organism before it has time to develop.


In this lab, we will be precipitating our own DNA. Precipitating DNA can be used by scientists and chemists to research genetics, practice cloning, test for traits or the probability of developing diseases, and also match DNA for forensics.

First we chew our cheeks to loosen cheek cells, then we swish around salene solution in our mouths. Then we spit the solution in a test tube. Next we add lysis buffer, which is a detergent, which breaks open the cell and nuclear membranes. This is required to access the DNA. Next we add protease, an enzyme that breaks down proteins. This is used because DNA is wrapped around proteins called histones, which let the DNA coil many, many times to reduce its size drastically. Removing these histones makes the DNA form into one large thread. The protease also breaks down unwanted molecules, like mitochondria, endoplasmic reticulum, and the golgi apparatus. Protease also destroys DNase, which is an enzyme that destroys DNA. If we want to collect the DNA, we want it to not be destroyed. Next we put the test tube in a water bath at 50 degrees Celcius, which catalyzes the reaction, mainly by helping the lysis buffer. Also many other bacteria will be killed at this temperature, and the growth of E. coli was slowed drastically.

Next we add cold ethanol, which cools the solution, which speeds the precipitation of DNA. After giving the DNA a few minutes to precipitate, we extract it from the solution with a pipette. We then put the DNA in a small container that is also a necklace piece, so we can have our own DNA in a super cool necklace.